- Mytilus Paper (defensin data) -
sr320 Jan 26, 2011
- Storer Fav Gene
June 3, 2011
Just a few last minute details
QPX Serine Protease accession number:
DQ925474
<span style="font-size: 13px;">atggacgtag tcaagcggat tcaaaaggcg agaccacgac ttgaagtaag tggcacccac
61 atgtatcatg ttgcagctgt ggcagtgcca aaaccatggg gactgaatcg agttgatcaa
121 cctgatctgc ctttagacca tacgtcgttc aagacgtcct acaccggtaa aaatgttacc
181 gtgtacatcc ttgataccgg aatttgggaa tcgcacggtg actatggcag tcgcgtgaga
241 cctggtgtta gtttcgtgaa aggggaggat tatgttatgg acagaaacgg gcatggcact
301 cattgtgcag gtactgctgt gggtacaaga tatggtgttg caaaagatgc ccaagttgtt
361 gggatcaaga tattgagtgc tgaagggtat ggaaacaccg tagatatcat caaaggtatt
421 gagtgggcag tcaacgatgc gcgatcaagg aataccccag gtgttctctc gctttctctt
481 ggtggcagcg cagaccctgt acttgacgca ggcgttgatg ccgccgccga tgcggggatg
541 ctggtcgcag tagcagctgg aaatgacaat ggcgatgcct gcaaaaagtc gccagcccga
601 gctgcccaag taatcacagt tggtgcaacg aatattgctg atcatcggtc ttacttttcc
661 aactggggca cttgcgtgaa tgtgttcgcc ccaggaacaa acatcatctc gtcatggatt
721 ggggcagaag gcgcagtaaa catgatcagt ggtacctcaa tggctacccc gcttgttgca
781 ggtgtgttgg cgactctact agagaaacac gatggagatg cggaggctgc aaaaatggac
841 atgtttactt tggttgcccg ggataaacta actgacgtca aagctagttc cccaaactgg
901 ttagtccaaa ctgcaagata atttggccta aaagttttaa ttctatttta ctgcaagact
961 gcaaaaaaaa aaaaaaa</span>
May 25, 2011
Sam ran a qPCR using LABY as a normalizing gene on the same samples as below. The results showed similar amplification as the serine protease gene. In both runs, the S-1, 7 ATCC and 81 strains did not work or show any amplification. The Cq data for the LABY gene can be seen here.
Worked on paper
May 18, 2011
The PCR from yesterday showed no contamination in the water samples and a positive result for the positive control, but the other values were a little "funky". The strains of QPX s-1, 81 and the new ATCC did not show any amplification.
The amplification of the old ATCC showed no obvious patterns, but the data will be analyzed quantitatively later.
Here is the amplification without the old ATCC, showing only the strains that did not work...
The melt peak shows no genomic DNA carryover.
From here on out, the old ATCC will be referred to as "68 ATCC" and they new ATCC will be referred to as "7 ATCC". The ATCC strain used in the first experiment from 3/11/2011 will be referred to as "22 ATCC". These numbers reflect how long the cells were growing for.
To see expression values for the data collected so far, see here.
May 17, 2011
Ran a PCR using the cDNA made on 5/12/2011. The positive control used was DNA isolated from ATCC 2/17/2011 QPX cultures on 3/11/2011.
Plate layout can be seen here.
Calculations:
Sso Fast Evagreen Supermix 12.5 x 25.3 = 316.25 ul
QPX SPB_F .5 x 25.3 = 12.65
QPX SPB_R .5 x 25.3 = 12.65
H2O 10.5 x 25.3 = 265.65
May 12, 2011
The concentrations of DNased RNA can be seen below.
The DNased RNA was changed into cDNA using the Promega M-MLV Reverse Transcription Protocol. I used .44ug of RNA in each sample. Full calculations can be seen here. (open using excel).
The next step is to run a PCR on the cDNA samples with a positive control and look for a difference in expression.
May 5, 2011 (Cinco de Mayo!)
Today I DNased the RNA samples from 5/4/2011 using the Turbo DNase Protocol. I used 2.5ug of RNA from every sample, which was limited by the low concentration of S-1 10C a. The volumes were then brought up to 50ul as usual. Calculations can for all of this can be seen here.
The next step is to run a PCR to ensure the DNasing worked and there is no genomic DNA carryover.
May 4, 2011
The cells were taken out of incubation from 10C and RT after 72 hours. Today, I isolated RNA from all of the samples using the Tri-Reagent Extraction Protocol. The RNA pellets were resuspended in 15ul of .1% DEPC treated water.
The concentrations can be seen below:
Note that the first 81 RT b is actually 81 RT a, there was just an error in labeling. Also, ignore the 5th entry labeled "old ATCC RT tissue b".
The samples were stored in -80C. The next step is to DNase the RNA tog et rid of any genomic DNA carry over.
April 26, 2011
To further explore my old experiment I am essentially doing two new experiments. In the first experiment, I am investigating how the presence of hard clam tissue changes the virulence or expression of serine protease at both 10C and Room Temperature. In the second experiment I seeing if the results from the previous experiment (relating temperature and virulence) hold true through three different strains of QPX: ATCC, S-1, and TD8-81.
I isolated the cells from the strains ATCC, S-1 and TD8-81 from 4/19/2011 (experiment 2) and from ATCC strain from 2/17/2011 (experiment 1). The cells were isolated from the mucous by mixing them with seawater, spinning in a centrifuge at 3000g for 10min, removing as much seawater from the top as possible and then repeating. The process takes about 5 cycles.
Experiment 1:
ATCC 10C with hard clam tissue (x2)
ATCC RT without hard clam tissue (x2)
The clam tissue was added after cells were isolated. The clam tissue was from a dead clam labeled "Hard Clam #23 1/9/09"
Experiment 2:
ATCC 10C (x2)
ATCC RT (x2)
S-1 10C (x2)
S-1 RT (x2)
TD8-81 10C (x2)
TD8-81 RT (x2)
-
April 19, 2011
Repeated the PCR from 4/15/2011 but running each sample twice. The annealing temperature was also raised to 60C to potentially get away from any dimers forming. Plate layout and cycling parameters can be seen here.
The results show no water contamination!
The amplification peak show some differences in expression between the 10C and Room Temperature. 10C shows amplification at an earlier cycle and ends with slightly more product.
The original results can be seen here.
The data above gives evidence that temperature does effect virulence in the serine protease gene. It seems that for colder temperatures, more serine protease is produced and virulence is increased. The next step would be to duplicate this experiment with multiple strains of QPX and see if the results transfer across strains. Another interesting topic could be to look into whether the presence of hard clam cells affect the the expression of serine protease. This experiment could be done using the same protocol as before only with several experimental groups. The samples would like like this:
Strain 1: 10C with HC tissue
Strain 1: 10C without HC tissue
Strain 1: Room Temperature with HC tissue
Strain 1: Room Temperature without HC tissue
Strain 2: 10C with HC tissue
Strain 2: 10C without HC tissue
Strain 2: Room Temperature with HC tissue
Strain 2: Room Temperature without HC tissue
Strain 3: 10C with HC tissue
Strain 3: 10C without HC tissue
Strain 3: Room Temperature with HC tissue
Strain 3: Room Temperature without HC tissue
Resulting in a total of 12 samples and three experimental groups.
April 15, 2011
Results from the qPCR showed dirty water samples, again. The graphs (seen below) are almost identical to the ones before. Next time, I will start with a new water aliquot and to make sure I am confident in these results I will use each cDNA sample twice. See original file here
NCBI has 28 ESTs for QPX. I ran a blast with all of them to see if they were associated with any gene and some resulted in significant results.
Blast 5, with name, gi|83278136|gb|DV942144.1|DV942144 has clearly already been sequenced because the E-value was extremely low.
Blast 12 with name gi|83278143|gb|DV942151.1|DV942151 was most similar to Phaeodactylum tricornutum, a diatom.
Blast 14, named gi|83278145|gb|DV942153.1|DV942153 is most similar to a ribosomal protein in Thalassoisira pseudonana, a marine diatom
Similar results came up for Blast 19, named gi|83278150|gb|DV942158.1|DV942158 so I made an allignment between the two and found out they were essentially the same sequence, but extended further in either direction.
April 13, 2011
Since the water samples were contaminated from the last PCR, I reran an identical PCR reaction.
Calculations:
Sso Fast Evagreen Supermix 12.5 x 5.5= 68.75ul
QPX SPB_F .5 x 5.5 = 2.75ul
QPX SPB_R .5 x 5.5 = 2.75ul
H2O 10.5 x 5.5 = 57.75ul
Plate layout and cycling parameters can be seen here.
April 6, 2011
The results from the PCR from 4/5/2011 showed contaminated water samples. The PCR will have to be rerun. However, the machine is not available today so I'll have to wait until I am in the lab next!
The amplification shows different levels of expression in the room temperature and 10C, which gives promising results for the next PCR. However, no conclusions can be drawn from the PCR because the water samples are contaminated.
The qPCR results in its entirety can be seen here.
April 5, 2011
Results from the qPCR from 4/1/2011:
There is no amplification other than the positive control, suggesting there is no genomic carry over in the RNA. One of the water samples shows some amplification towards the end of the cycles and this can be better analyzed in the melt peak.
Since the water sample does not have a melt peak at the same temperature as the positive control, it is not contaminated. There is no activity by either the 10C or Room Temperature samples, so it can be concluded that there is no genomic carry over.
The next step is to make cDNA from the RNA samples using the Promega M-MLV protocol. Concentrations of the two samples can be seen below:
*Ignore the first Room Temperature sample, there was an air bubble in the spectrophotometer.
The calculations for the reverse transcription reaction can be seen here. Everything else followed the M-MLV protocol.
Ran a PCR using the resulting cDNA and the ATCC DNA for a positive control.
Calculations:
Sso Fast Evagreen Supermix 12.5 x 5.5= 68.75ul
QPX SPB_F .5 x 5.5 = 2.75ul
QPX SPB_R .5 x 5.5 = 2.75ul
H2O 10.5 x 5.5 = 57.75ul
Plate layout and cycling parameters can be seen here.
April 1, 2011
Upon second look at the PCR results from 3/30/2011, the PCR reaction did not work, which is obvious because the fluorescence came up way too early. So, I reran the PCR using the DNased RNA from 3/31/2011 and DNA labeled ATCC, which was isolated on 3/11/2011, as a positive control. Including a positive control and re-running the reaction will give us results that hopefully show that there is no genomic carryover.
Calculations:
Sso Fast Evagreen Supermix 12.5 x 5.5= 68.75ul
QPX SPB_F .5 x 5.5 = 2.75ul
QPX SPB_R .5 x 5.5 = 2.75ul
H2O 10.5 x 5.5 = 57.75ul
Plate layout and cycling parameters can be seen here.
March 31, 2011
Results from 3/30/11 showed some amplification and genomic DNA carryover. The RNA from the 10C sample showed a higher amplification than the Room Temperature. To get rid of the genomic DNA, the RNA was DNased using the Turbo DNAse protocol. The reaction volume used was 50ul. Calculations can be seen below:
5ug/.19845ug/ul = 25.195 ul of Room Temperature RNA
5ug/ .25698 ug/ul = 19.46ul of 10C RNA
Next, I need to run a qPCR on this to make sure the reaction worked and there is no genomic DNA.
March 30, 2011
Ran a qPCR on the RNA isolated on 3/14/2011. The goal of this was to determine if there is any genomic DNA leftover.
Calculations:
Sso Fast Evagreen Supermix 12.5 x 4.4 = 55ul
QPX SPB_F .5 x 4.4 = 2.2ul
QPX SPB_R .5 x 4.4 = 2.2ul
H2O 10.5 x 4.4 = 46.2ul
Cycling Parameters and plate layout can be seen here.
March 14, 2011
To recover cells from the seawater they were re-suspended in, I spun the samples at 3000rcf for 10 minutes and then removed the aqueous layer to leave the pellet of cells. The samples that were incubated at 10c were spun at 10C and the room temperature samples were spun at room temperature. The two samples that were in each treatment group were combined, so we were left with one 10C sample and one Room Temperature sample. Once the cells were recovered, I used the Tri-Reagent RNA extraction protocol to isolate RNA. Each sample of RNA was re-suspended in 50ul DEPC treated water. The concentrations of each of the two samples can be seen below.
The concentrations are high, but the 260/280 values are not great. Ideally, they should be between 1.9-2.0, but since there is not a lot of information on QPX, there could be something in its biology that makes these values lower. The next step is to run a qPCR to look for any genomic DNA carry over.
March 11, 2011
Isolated cells from four alloquats of ATCC 2/17/2011 QPX cultures. The cells were isolated from the mucous by mixing them with seawater, spinning in a centrifuge at 3000g for 10min, removing as much seawater from the top as possible and then repeating. The process takes about 5 cycles. Once the cells were isolated, they were re-suspended in 14ml of sterilized seawater. 3ml was divided into 4 flasks. At 3:40pm, two of these flasks were left out in room temperature and the other two were incubated at 10C. They will be left there over the weekend, or approximately 70 hours.
The next step is to isolate RNA.
March 9, 2011
Now that we know the Serine Protease genes for QPX genes "work", we can set up an experiment to understand differences in expression of the serine protease gene in response to different environmental cues. Serine protease serves as a measure of virulence and by exposing it to different temperatures, we can get insight to how virulence may change in response to climate change.
Basic Procedure:
Step 1: Isolate cells from mucous using sea water technique
Step 2: Divide cells into 4 alloquats and add antibiotics
Step 3: Incubate three samples at 10C and the other two room temperature, creating two treatment groups "cold" and "room temperature".
Step 4: Isolate RNA from both treatment groups, using the TriReagent protocol
Step 5: Transcribe RNA to cDNA using the Promega M-MLV protocol
Step 6: Run a qPCR on all samples and look for differences in expression.
Details I need to figure out:
How long to incubate the samples
Will a temperature of 37C kill off the cells
Should there be more than 2 treatment groups
March 4, 2011
Because the water samples were either contaminated or showed a primer dimer, Sam and I both reran the reaction. In theory, if we both get a soft band, than the product we can see is a primer dimer. If I get a band, but he doesn't, than the contamination is likely due to an error in technique.
2x Taq mm 12.5 x 4.4 = 55ul
QPX SPB_F .5 x 4.4 = 2.2ul
QPX SPB_R .5 x 4.4 = 2.2ul
H2O 10.5 x 4.4 = 46.2ul
Cycling Parameters:
95 for 15sec
50 for 15sec
72 for 30sec
for 40 cycles
As you can see, the water in my samples turned up with only soft bands that were not the same molecular weight as the product, which indicates a primer dimer. One of Sam's water samples was contaminated.
March 2, 2011
The DNA from 2/24/2011 formed a pellet after sitting at room temperature. After discarding the pellet, I found the concentration of the DNA in aqueous solution. Keep in mind that 1 and 2 aren't from different samples, they were the same sample separated into two aliquots to make extraction easier.
Set up a PCR reaction with these samples and the QPX SPB 10uM primers.
2x Taq mm 12.5 x 4.4 = 55ul
QPX SPB_F .5 x 4.4 = 2.2ul
QPX SPB_R .5 x 4.4 = 2.2ul
H2O 10.5 x 4.4 = 46.2ul
Cycling Parameters:
95 for 15sec
50 for 15sec
72 for 30sec
for 40 cycles
Ran sample out on a 1% agarose gel with ethidium bromide, but there was contamination in the second water sample.
Reran the PCR with the same reaction conditions and cycling parameters in attempt to get clean water samples.
Set up a PCR reaction with these samples and the QPX PBR primers.
2x Taq mm 12.5 x 4.4 = 55ul
QPX SPB_F .5 x 4.4 = 2.2ul
QPX SPB_R .5 x 4.4 = 2.2ul
H2O 10.5 x 4.4 = 46.2ul
Cycling Parameters:
95 for 15sec
50 for 15sec
72 for 30sec
for 40 cycles
Ran the PCR products out on a 1% agarose gel with ethidium bromide. Results showed two contaminated waters this time.
February 24, 2011
The samples from yesterday were cloudy and didn't precipitate well into solution, so I tried to precipitate with ethanol using this protocol:
Ethanol Precipitation (DNA)
Add 1/10 volume of 3M NaOAC (pH=5.2) to sample.Invert tube several times to mix.
Add 2 volumes of ice cold 100% EtOH to sample.
Invert tube several times to mix.
Incubate @ -20C for at least 30 mins. Can go longer if desired.
Centrifuge at highspeed (~16,000g) @ 4C for 30 mins to pellet DNA.
Discard supernatant.
Wash pellet with 70% EtOH.
Spin 5 mins. at highspeed (`16,000g) @ 4C to repellet DNA.
CAREFULLY remove supernatant.
Air dry pellet for 5-10 mins.
Resuspend DNA pellet in appropriate volume of water or Qiagen EB (10mM Tris-HCl, pH=7.5)
I resuspended the DNA pellet into TE, but it didn't dissolve as it should have. I transferred half of the sample to a different snap cap tube and brought both volumes up to 1mL. However, there was still some precipitate and it was slightly cloudy. Put the samples on Sam's desk for him to take look.
February 23, 2011
Isolated DNA from QPX samples labeled ATCC 12/23/2010. First the cells needed to be cleaned to eliminate the mucus and make the cells accessible. The DNA was isolated using the DNAzol protocol and was resuspended in 250ul of NaOH.
Prepared for a DNA extraction using the DNAzol protocol on Oyster Samples for next time.
1. LYSIS \ HOMOGENIZATION: 1 ml DNAzol + 25 - 50 mg tissue, 107 cells or 0.1 ml liquid sample.
2. CENTRIFUGATION (Optional): 10,000 g x 10 minutes.
3. DNA PRECIPITATION: lysate + 0.5 ml 100% ethanol.
4. DNA WASH: 1 ml 75% ethanol (2x).
5. DNA SOLUBILIZATION: 8 mM NaOH or water.
1. LYSIS / HOMOGENIZATION
A. TISSUES. Homogenize tissues in a hand held glass-Teflon homogenizer. Use a loosely fitting homogenizer, with a tolerance greater than 0.1-0.15 mm. Homogenize 25-50 mg tissue in 1 ml of DNAzol by applying as few strokes as possible. Typically, 5-10 strokes are required for complete homogenization. Small amounts (5-10 mg) of soft tissues, such as spleen or brain can be dispersed and lysed by repetitive pipeting with a micropipette. Store the homogenate for 5-10 minutes at room temperature.
(Optional)
Sediment the homogenate for 10 minutes at 10,000 g at 4-25 C. Following centrifugation, transfer the resulting viscous supernatant to a fresh tube.
This step removes insoluble tissue fragments, partially hydrolyzed RNA and excess polysaccharides from the lysate/homogenate. It is required only for the isolation of DNA from tissues such as liver, muscles and most plant tissues containing a large amount of cellular and/or extracellular material and is also recommended for the isolation of RNA-free DNA.
3. DNA PRECIPITATION
Precipitate DNA from the lysate/homogenate by the addition of 0.5 ml of 100% ethanol per 1 ml of DNAzol used for the isolation. Mix samples by inverting tubes 5-8 times and store at room temperature for 1-3 minutes. Make sure that DNAzol and ethanol mix well to form a homogenous solution. DNA should quickly become visible as a cloudy precipitate. Remove the DNA precipitate by spooling with a pipette tip. Swirl the DNA onto the tip and attach it to the tube wall near the top of the tube by gently sliding the DNA off the tip. Alternatively, transfer the DNA to a clean tube. Store the tubes upright for about 1 minute and remove from the bottom of the tubes the remaining lysate/homogenate.
Degraded DNA and small quantities of DNA (< 15 µg) do not spool onto a pipette tip. In this case, sediment the precipitated DNA by centrifugation at 5,000 g for 5 minutes at 4 -25 C.
4. DNA WASH
Wash the DNA precipitate twice with 0.8 - 1.0 ml of 75% ethanol. At each wash, suspend the DNA in ethanol by inverting the tubes 3 - 6 times. Store the tubes vertically for 0.5 - 1 minutes to allow the DNA to settle to the bottom of the tubes and remove ethanol by pipetting or decanting.
If necessary, sediment the DNA pellet at 1,000 g for 1 - 2 min at 4 - 25 C. To further remove contaminants when isolating DNA from tissues, the first ethanol wash can be replaced with wash in a solution containing 70% DNAzol and 30% ethanol.
February 22, 2011
Got the results of the qPCR from 2/16/2011. The results are the same, but with less strange samples.
Amplifiation Curve:
As you can see, the only sample that showed significant amplification was pooled cDNA.
Again, the melt curve shows a peak for a lower temperature than the peak for pooled cDNA and MA6. This could be because of a primer dimer since nearly every sample has that mini-peak.
This results, combined with the other show that this primer, Big Def, does not seem to be present in nearly all of these samples. The pooled cDNA, which was samples taken from different hard clams in February 2010, did show expression. The MA6 sample showed some expression as well, but in a small quantity.
February 16, 2011
Reran the PCR from 2/14/2011 with the same samples and cycling parameters.
Calculations:
2x qPCR MM 12.5 x 37.4 = 467.5ul
Big Def_1 F .5 x 37.4 = 18.7ul
Big Def_1 R .5 x 37.4 = 18.7ul
Water 10.5 x 37.4 = 392.7ul
Plate layout and cycling parameters can be seen here.
Researched evolutionary conservation of the Big Defensin gene through NCBI and other databases.
Conservation of an unidentified defensin from Crassostrea giga (oyster):
http://www.jbc.org/content/281/1/313.full.pdf
Talks about y(gamma)-core, which I'm assuming is the core promoter of several genes, including big defensin. Core promoters are sequences upstream from the gene which initiates transcription. Since they are small and essential in transcription, they are highly conserved.
http://www.pnas.org/content/101/19/7363.full
Big Defensin found in Horseshoe crab. Has an analysis and conservation of insect defensin:
http://onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.1996.tb52963.x/pdf
Comparing Big Defensin between mollusks: scallops, horseshoe crab and lancelets:
http://www.sciencedirect.com.offcampus.lib.washington.edu/science?_ob=ArticleURL&_udi=B6T9R-4JMVHGX-4&_user=582538&_coverDate=01/31/2007&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor
=&view=c&_searchStrId=1644520962&_rerunOrigin=scholar.google&_acct=C000029718&_version=1&_urlVersion=0&_userid=582538&md5=
2129647a287f0f667e2b112ce3c4222c&searchtype=a
Proposition of the 3-dimension shape of Defensin and what each terminus looks likes molecularly:
http://pubs.acs.org/doi/abs/10.1021/bi900756y
Motifs in defensin structures:
http://www.nature.com/ni/journal/v6/n6/full/ni1206.html
When researching, it became clear that there are two main methods to classifying defenins: alpha-defensin and beta-defensin. They are classified based on tri-disulfide linkages in the peptides. Beta-defensins are slightly larger and differ in the position and arrangement of 3 disulfides. Big defensin is usually classified under Beta because one of its termini is similar to the beta-defensin terminus
February 15, 2011
Results from the qPCR on 2/9/2011 showed very little amplification in the samples, other than the cDNA. This is somewhat expected as the last 2 conventional PCRs with this gene show no amplification other than the pooled cDNA. However, based on the results from 1/11/2011, we would expect to see products in the samples CA, CA5, MA4 and MA5 as well. This was not the case.
Amplification Curve:
As you can see, the only sample that showed significant amplification was the pooled cDNA. However, there is a another sample rising much less steep and earlier. There was no sample in the well this corresponds to, so I am not sure what happened.
Amplification Curve (log scale):
The log scale of amplification makes it easier to see the empty well that showed amplification. Other than the pooled cDNA, there were two other samples that stood out; MA9 and CA9. While the samples didn't show very much amplification, it was expressed at around the same time, so it could be a similar product. The melting curve can tell us more on that subject.
Melting Curve:
The only strong peak on the melting curve is at the pooled cDNA. There seems to be some product in MA9 (which supports what I was saying above), however it comes too early for the expected product. There looks at is if there is some product at the correct melting temperature under the pooled cDNA curve with the samples MA1 and MA6. These samples have never shown any product and it is still very very slight, so no conclusions can be drawn from this. The MA10 sample has the opposite pattern of a normal melting curve and I have no explanation for this, it is very strange.
To see original data file for qPCR results, click here.
Next: Since there are several (in Sam's words) "funky" samples here, the best option would be to repeat the qPCR and see if things change.
February 9, 2011
Set up a qPCR reaction using all the samples from 1/5/2011 to explore differences in expression for the Big Defensin gene. The samples are taken from three sites and the samples labeled MAX were exposed to QPX. The Promega 2x qPCR Mastermix was used in setting up these reactions.
Calculations:
2x qPCR MM 12.5 x 37.4 = 467.5ul
Big Def_1 F .5 x 37.4 = 18.7ul
Big Def_1 R .5 x 37.4 = 18.7ul
Water 10.5 x 37.4 = 392.7ul
Plate layout and cycling parameters can be seen here.
On a different note, I ran a conventional PCR on serine protease primers given from Sam titled QPX SPB FW 10uM. The template used was cDNA samples from Sam labeled ATCC 4/22/08. The PCR was run with the following cycling parameters:
95 fo 15sec
55 for 15sec
72 for 30sec
for 40 cycles
Calculations:
2x Taq mm 12.5 x 3.3 = 41.25ul
QPX SPB FW_F .5 x 3.3 = 1.65ul
QPX SFB FW_R .5 x 3.3 = 1.65ul
H2O 10.5 x 3.3 = 34.65 ul
After running the PCR products out on a gel, there appears to be no amplification in the cDNA or in the waters so either this gene was not expressed in these samples or the primers did not "work"
February 8, 2011
Ran the PCR results from 2/04/2011 out on a gel. The same order (Ladder, CA1, CA2, MA1, MA6, cDNA pooled, water, water) was used in loading the gel. The results were consistent with the previous PCR results from 2/1/2011, showing a band only in the positive control cDNA samples. This band is around the expected sample size
February 4, 2011
Apparently the gel from 2/22011 showed a contamination in the second water sample. So, today I reran the PCR to try and get clean water samples and consistent results. The same samples were used.
Calculations:
2x Taq mm 12.5 x 7.7 = 96.25 ul
MM_BigDef1_F .2 x 7.7 = 3.85 ul
MM_BigDef1_R .2 x 7.7 = 3.85 ul
H2O 11.1 x 7.7 = 80.85 ul
Cycling Parameters:
95 for 15sec
50 for 15sec
72 for 3:00min
for 40 cycles
Made a 1% agarose gel with ethidium bromide and stored in -20.
February 2, 2011
Reran the PCR from 2/1/2011 in attempt to duplicate the results. The same calculations and cycling parameters were used.
Calculations:
2x Taq mm 12.5 x 7.7 = 96.25 ul
MM_BigDef1_F .2 x 7.7 = 3.85 ul
MM_BigDef1_R .2 x 7.7 = 3.85 ul
H2O 11.1 x 7.7 = 80.85 ul
Cycling Parameters:
95 for 15sec
50 for 15sec
72 for 3:00min
for 40 cycles
The samples were run on a 1% agarose gel with ethidium bromide in the same order as before (CA1, CA2, MA1, MA6, pooled cDNA, water, water). As you can see, the results were consistent showing no bands in any samples other than the positive control, pooled cDNA.
February 1, 2011
Ran the PCR products out on a 1.5% agarose gel with ethidium bromide. These samples are the same as the ones labeled in red below, however, this time a product was seen in the positive control. The order of the wells, CA1, CA2, MA1, MA6, pooled cDNA, water, water.
January 28, 2011
Ran the PCR products from 1/26/2011 and 1/21/2011 out on a 1.5% agarose gel with ethidium bromide.
The black font are the samples from 1/21/2011 where the primer concentration was decreased in attempt to get rid of the primer dimer make the product bands more clear. The results showed a more faint, but still very present primer dimer and no cDNA bands.
The red font shows the PCR samples from 1/26/2011 that used a new Big Defensin primer (order; CA1, CA2, MA1, MA6, positive cDNA control, water, water). A positive cDNA control was used to see if the primer "worked", but no bands were seen. It is possible, however, that big defensin was not expressed in any of these samples.
Reran the PCR for the samples in Red, with the MMBigDef1 Primer in attempt to get different results. The annealing temperature was changed to 50C.
Calculations:
2x Taq mm 12.5 x 7.7 = 96.25 ul
MM_BigDef1_F .2 x 7.7 = 3.85 ul
MM_BigDef1_R .2 x 7.7 = 3.85 ul
H2O 11.1 x 7.7 = 80.85 ul
Cycling Parameters:
95 for 15sec
50 for 15sec
72 for 15sec
for 40 cycles
January 26, 2011
Ran a PCR using the new primers. The samples I used were the same CA1, CA2, MA1 and MA6, plus two water samples and a pooled cDNA I had from a previous hard clam experiment.
Calculations:
2x Taq mm 12.5 x 7.7 = 96.25 ul
MM_BigDef1_F .2 x 7.7 = 3.85 ul
MM_BigDef1_R .2 x 7.7 = 3.85 ul
H2O 11.1 x 7.7 = 80.85 ul
Big defensin, a protein associated with antimicrobial defense, consists of 79 amino acids. The protein has two functional active sites where execute different antimicrobial activities. The A-terminus deals with Gram-positive bacteria while the C-terminus deals with Gram-negative bacteria. (Gram-positive/negative is a way to classify bacteria based on the composition of the cell walls). Defensin is most similar to Alpha-defensin3 in humans and other primates.
A blastn of the sequence of Mercenaria mercenaria big defensin (accession number) resulted in only 4 results with no significance. More on this later.
January 21, 2011
Ran another PCR using the same samples from 1/19. This time, the volume of primer added was decreased to .2ul to attempt to get rid of the primer dimer band. The cycling parameters went back to the original parameters (from 1/7) because that is where the most amplification was seen.
Calculations:
2x Taq mm 12.5 x 6.6 = 82.5ul
MM_BigDef_F .2 x 6.6 = 1.32 ul
MM_BigDef_R .2 x 6.6 = 1.32 ul
H2O 11.1 x 6.6 = 73.26 ul
Cycling Parameters:
95 for 15sec
55 for 15sec
72 for 15sec
for 40 cycles
Designed new primers for the same Big Defensin gene with the accession number GO915266. The primers are under the database named as MM_BigDef1_F and MM_BigDef1_R
January 19, 2011
Reran PCR from the same samples (CA1, CA2, MA1, MA6) since the gel from 1/13 was inconsistent with the gel from 1/11. The same calculations and cycling parameters were used:
2x Taq mm 12.5 x 6.6 = 82.5ul
MM_BigDef_F .5 x 6.6 = 3.3 ul
MM_BigDef_R .5 x 6.6 = 3.3 ul
H2O 10.5 x 6.6 = 69.3 ul
Cycling Parameters:
95 for 15sec
55 for 15sec
72 for 15sec
for 40 cycles
Ran PCR products out on a 1.5% agarose gel with ethidium bromide. This samples in wells from left to right are ladder, water, water, CA 1, CA2, MA1, MA6. MA6
This shows that only one sample, MA6, had a band other than the primer dimer. Since this is inconsistent with the results from 1/11 and 1/13, I reran the PCR for the third time. However, the papers in which the primers came from (see here), used an annealing temperature of 60C, instead of 55. So this trial was run with a higher annealing temperature in hopes to decreaes the thick primer dimer and increase the correct band.
Calculations:
2x Taq mm 12.5 x 6.6 = 82.5ul
MM_BigDef_F .5 x 6.6 = 3.3 ul
MM_BigDef_R .5 x 6.6 = 3.3 ul
H2O 10.5 x 6.6 = 69.3 ul
Cycling Parameters:
95 for 15sec
60 for 15sec
72 for 15sec
for 40 cycles
Ran the PCR products out on a 1.5% agarose gel with ethidium bromide. The samples in wells from left to right are ladder, water, water, CA1, CA2, MA1, MA6. There are no bands on any of the samples, other than the primer dimer. This does not support any of the previous data.
January 13, 2011
Ran the PCR products from 1/12/11 out on a 1.5% agarose gel with ethidium bromide.
. There were multiple band for all of them this time, including the water samples. From left to right, the samples are ladder, CA1, CA2, MA1, MA6, Water, Water.
January 12, 2011
Reran a PCR on only a few samples. Samples used were CA1, CA2, MA1 and MA6. CA2 and MA6 had the second band and CA1 and MA1 did not. The same primers and cycling parameters were used.
2x Taq mm 12.5 x 6.6 = 82.5ul
MM_BigDef_F .5 x 6.6 = 3.3 ul
MM_BigDef_R .5 x 6.6 = 3.3 ul
H2O 10.5 x 6.6 = 69.3 ul
Cycling Parameters:
95 for 15sec
55 for 15sec
72 for 15sec
for 40 cycles
The expected size of this PCR product is 295 base pairs
January 11, 2011
Ran a gel on the PCR products from 1/7/11. The products were run on a 1.5% agarose gel. The first row contains (from well 1-20) ladder, water, water, CA 1-CA10, MA1-MA7. The second row contains MA7-MA10 and MAX1-MAX10.
Each sample resulted in a band, however it is probably due to the primer dimer. The PCR product is probably the second band, as seen in CA2, CA6, MA6 and MA7. The PCR product size is not listen in the primer database, but it is probably small, given these results.
January 7, 2010
Ran a PCR on the cDNA samples generated on 1/5/11. The Primer used was MM_BigDef.
Calculations:
2x Taq mm 12.5 x 35.2 = 440ul
MM_BigDef_F .5 x 35.2 = 17.6 ul
MM_BigDef_R .5 x 35.2 = 17.6 ul
H2O 10.5 x 32.5 = 369.6 ul
Cycling Parameters:
95 for 15sec
55 for 15sec
72 for 15sec
for 40 cycles
January 5, 2010
Translated RNA to cDNA using the Promega M-MLV Protocol. from Dave's Hard Clam Samples. See here for amount of RNA used and calculations.
(transcribed!) be sure to try to get all data entered / linked the day of -
Samples were stored @ -20C in "Lexie's Samples" box. -
December 10, 2010
Ran a PCR using the SPI Primers designed from November 9, 2010 (ID numbers: Vp_SPI_allign_F and Mm_SPI_allign_R). The goal of this PCR was to see if the primers "worked", so pooled cDNA was used as a template. The PCR products were run on a 1% gel with ethidium bromide and were run on the same gel as the digests from December 7, 2010. Both digests were run, Sample A had 1ul of enzyme and Sample B had no enzyme. Both samples had 20ul of cDNA was the concentration of 26.41ng/ul.
Results from gel:
Lane 1: Ladder
Lane 2: SPI Primers with pooled cDNA
Lane 3: Water
Lane 4: Water
Lane 5: Restriction Digest w/ enzyme (A)
Lane 6: Restriction Digest w/o enzyme (B)
The SPI Primers showed no amplification with the pooled cDNA, likely showing that they did not "work". However, the product from the primers would be a product under 100bp long, so it may be too small for us to see. To determine which, a qPCR should probably be done.
The restriction digest, A, which included the HPAII enzyme seemed to have worked normally. However, sample B was a little strange and should not have shown that much amphlificaiton.
December 7, 2010
Spectrophotometer results DNA samples from November 29, 2010:
Obviously, these results are very poor. There is not a high concentration of DNA, especially in sample 4. The blank used was 100ul of NaOH and 1ul of HEPES. The 260/280 values are bad for all of them, but, again, sample 4 is crazy. The 260/230 values are also awful, but close to each other which makes me think they are more reliable.
Made HPA11 enzyme reactions using sample #2.
Reaction A:
20 ul DNA
5 ul 10x Buffer
1ul HPAII Enzyme
24 ul H2O
Reaction B:
20 ul DNA
5 ul 10x Buffer
25ul HPAII Enzyme
There was not enough of the HPAII enzyme left, so Reaction B (containing everything but the enzyme) is stored in my -20box and reaction A is placed in 37C heat bath overnight.
December 2, 2010
Entered gigas data from May and July field samples into the Cgigas_FieldData database.
November 29, 2010
Isolated DNA from oyster samples labeled "Untreated Controls 4/15/10 AD". There was no specific tissue taken, the tissue was simply taken from any part of the oyster. The DNAzol protocol listed below was used for all six samples. The isolated DNA was suspended in 100ul of 8mM NaOH and 1ul of HEPES.
Note: The samples themselves are labeled with their number on the shell, however the last oyster shell could not be written on, so sample 6 is the flat oyster without a label.
November 12-17, 2010
Designed primers by looking at alignments of similar organisms and looking for highly conserved regions. The best primers found were the ones designed on 9/9/2010. These primers were Forward: GTTATAAGCTACGGATC and Reverse: TGACTGCAAGACAA, which is the complementary base pair of the reverse primer below. These primers will be ordered and then used in a PCR to determine the 5' end of the Mercenaria SPI gene.
Prepared for a DNA extraction using the DNAzol protocol on Oyster Samples for next time.
1. LYSIS \ HOMOGENIZATION: 1 ml DNAzol + 25 - 50 mg tissue, 107 cells or 0.1 ml liquid sample.
2. CENTRIFUGATION (Optional): 10,000 g x 10 minutes.
3. DNA PRECIPITATION: lysate + 0.5 ml 100% ethanol.
4. DNA WASH: 1 ml 75% ethanol (2x).
5. DNA SOLUBILIZATION: 8 mM NaOH or water.
1. LYSIS / HOMOGENIZATION
A. TISSUES. Homogenize tissues in a hand held glass-Teflon homogenizer. Use a loosely fitting homogenizer, with a tolerance greater than 0.1-0.15 mm. Homogenize 25-50 mg tissue in 1 ml of DNAzol by applying as few strokes as possible. Typically, 5-10 strokes are required for complete homogenization. Small amounts (5-10 mg) of soft tissues, such as spleen or brain can be dispersed and lysed by repetitive pipeting with a micropipette. Store the homogenate for 5-10 minutes at room temperature.
(Optional)
Sediment the homogenate for 10 minutes at 10,000 g at 4-25 C. Following centrifugation, transfer the resulting viscous supernatant to a fresh tube.
This step removes insoluble tissue fragments, partially hydrolyzed RNA and excess polysaccharides from the lysate/homogenate. It is required only for the isolation of DNA from tissues such as liver, muscles and most plant tissues containing a large amount of cellular and/or extracellular material and is also recommended for the isolation of RNA-free DNA.
3. DNA PRECIPITATION
Precipitate DNA from the lysate/homogenate by the addition of 0.5 ml of 100% ethanol per 1 ml of DNAzol used for the isolation. Mix samples by inverting tubes 5-8 times and store at room temperature for 1-3 minutes. Make sure that DNAzol and ethanol mix well to form a homogenous solution. DNA should quickly become visible as a cloudy precipitate. Remove the DNA precipitate by spooling with a pipette tip. Swirl the DNA onto the tip and attach it to the tube wall near the top of the tube by gently sliding the DNA off the tip. Alternatively, transfer the DNA to a clean tube. Store the tubes upright for about 1 minute and remove from the bottom of the tubes the remaining lysate/homogenate.
Degraded DNA and small quantities of DNA (< 15 µg) do not spool onto a pipette tip. In this case, sediment the precipitated DNA by centrifugation at 5,000 g for 5 minutes at 4 -25 C.
4. DNA WASH
Wash the DNA precipitate twice with 0.8 - 1.0 ml of 75% ethanol. At each wash, suspend the DNA in ethanol by inverting the tubes 3 - 6 times. Store the tubes vertically for 0.5 - 1 minutes to allow the DNA to settle to the bottom of the tubes and remove ethanol by pipetting or decanting.
If necessary, sediment the DNA pellet at 1,000 g for 1 - 2 min at 4 - 25 C. To further remove contaminants when isolating DNA from tissues, the first ethanol wash can be replaced with wash in a solution containing 70% DNAzol and 30% ethanol.
Once DNA is extracted it is going to be exposed to restriction enzymes via the protocol:
General Restriction Digest Protocol:
- Extract DNA from acid shocked/control juveniles (tails)
- Run restriction digest with HPAII/MSPI
- Run products on gel
- PCR for methylation fingerprinting
For HPAII:
1 ug DNA (1/concentration x .001)
5 uL 10X Buffer 1
1 uL HPAII enzyme
Water up to 50uL
For MSPI:
1 ug DNA
5 uL 10X Buffer 4
1 uL MSPI enzyme
Water up to 50uL
November 9, 2010
Designed primers that were intended to understand the first part of the Mercenaria sequence. To do this, an alignment of ACU83222 and Mercenaria serine protease inhibitor genes at the nucleotide level. This can be seen below. GQ384398 is the accession number for ACU83222 at the nucleotide level. The goal was to get the first part of the sequence, and thus design primers so they would anneal to the "missing" 40 base pairs. The primers developed were intended to just isolate that region, rather than amplifying the entire gene.
The two primers I found were Forward: GTTATAAGCTACGGATC and Reverse: ACGGACCATCTGTT
However, one of these needs to be reversed into its complementary base. The forward primer was extracted from base pairs 4-20 of the sequence GQ384398, which is the nucleotide sequence of ACU83222. The reverse primer was taken from base pairs 44-32 of the consensus identity in the alignment seen above. Base Pairs 52-41 are from Mercenaria and base pairs 39 and 40 are from GQ384398. See here for the thought process and diagram of these primers.
A blastn was done on the base pairs of the consensus identity from the alignment above (first 41 base pairs of GQ384398). The goal of this was to find highly conserved areas, so we can design primers and be more confident they will be seen in the Mercenaria sequence. As seen below, no significant results were found. All results had very low E-values (other than the first one, which is just the sequence itself), and most of the species were not closely related to Mercenaria.
Note: the alignment of GQ384398 and Mercenaria as well as the .jpeg of the blastn for GQ384398 is saved in the main folder of My Dropbox. I had trouble with the networks today, but will fix next time I am in.
November 4, 2010
The most interesting result from the blastp of the Mercenaria translation was the alignment of ACU8322. This gene is a serine protease inhibitor from Venerupis philippinarium, a salt-water clam.
Since the Mercenaria gene does not have a start codon, it is clear that there is more to the sequence than what is shown. This alignment offers a possible sequence to be added to the begining of the Mercenaria SPI gene to extend and complete it. The XP_002368739 alignment, shown below, offers another explanation. This sequence would suggest 12 additional base pairs, and the previous one would only suggest 9. This gene is a serine protease inhibitor fromt eh species Toxoplasma gondii, a parasitic protozoa.
November 3, 2010
The initial hits from Mercenaria blastn showed poor results. The top 7 results, all with e-values in between 3.46E-11 and 1.47E-9, were only 43-47 base pairs long and the significant overlap happened in the poly-A tail, which was well outside the open reading frame. To fix this, the ORF was run through a blastx and blastn to see if results were at all significant. The blastn resulted in poor E-values (the top ten ranged from 1.93E-4 to 4.24E0). The blastx of the ORF resulted in better e-values and some interesting result. Results of the blast can be seen below. Of these results, there were more serine protease inhibitor proteins than any other protein, confirming that this is the serine protease gene. There were several kazal-type serine protease inhibitor genes listed with a high e-value. Kazal-type serine protease inhibitors in humans is associated with pancreatitis and is said to have something to do with immunity. Kazal-type is a family of serine protease inhibitors that contain at least one domain containing the characteristic three disulfide bridges. These domains and the similar quaternary structure are highly conserved through 100+ types of kazal-type SPIs studied. Since these are highly conserved, this might suggest that the gene we are looking at is a kazal-type SPI.
October 29, 2010
Researched the Mercenaria mercenaria Serine Protease Inhibitor Gene using genious to gain information based on how many organisms have similar gene sequences and how similar they are. Information was gathered for analysis at a later date. The main techniques used were blastx and blastn of the gene sequence and looking at the resulting organisms and comparing them to Mercenaria mercenaria.
October 27, 2010
An important role of protease inhibitors in an involvement in host defense. This is usually done by an organisms protease inhibitors directly inhibiting the protease of the pathogen. In theory, this would lead to more expressed SPI in the clams exposed to QPX and opposed to the control. The first pure protease inhibitors were extracted in the plasma of mollusks, but they have been found in both gill and hemolymph tissues from Mercenaria mercenaria. In humans, serine protease inhibitors have roles outside of protease inhibition, some are found to stop metastasis in breast and prostate cancer. Another type of serine protease inhibitor is involved in chromatin remodeling during mitosis/meiosis in red blood cells. In prokaryotes, serine proteases also serve as extra resistance to heat denaturation. The structure of serine proteases is always 3-Beta Pleated Sheets and 8-9 alpha helicies and has two active sites in which inhibitors or substrate could enter.
October 25, 2010
Explored the Mercenaria Mercenaria SPI Gene by comparing it to other species using the PubMed BLAST tool. This gene showed no overlap from the SPI genes of the species Homo sapiens, Drosphilia melanogaster, Mus musculus, Danio rerio. The common name for these species are humans, common fruit fly, house mouse and zebrafish. These species are phylogenetically very far from Mercernaria mercenaria and it makes sense that there would be no significant overlap. A blastx was done with the Mercenaria Mercenaria SPI gene and the results can be seen here. As can be seen, there is a wide range of results, the top two being Toxoplasma gondii, a parasitic protozoa, and Venerupis philippinarum, another species of saltwater clam.
October 24, 2010
A serine protease is an enzyme that cuts a peptide bond in developing proteins. It is named from serine, an amino acid that is always found in the active site. Serine Protease Inhibitors, also called Serpins, inhibit the activity of serine proteases by mimicking the shape and structure of the substrate. When the serpin binds to the serine protease, it becomes cut by the protease, leading to a covalent bond between the two molecules. When the serpin and serine protease bind to each other, the serpin changes shape drastically which signals the organism to move it to be destroyed.
October 15, 2010
PCR from October 11, 2010 resulted in positive results for the HSP70 gene, but not the Efb for the samples. The pooled cDNA showed positive amplification for both genes, and all water samples were clean. A possible explanation for this would be that the Reverse Transcription reaction did not work and HSP70 only showed amplifiication because of DNA amplification. This would show up in the HSP70 gene and not the Efb if the Efb gene had introns that added too many base pairs in between primers, which would not be amplified under the cycling parameters.
HSP70 is in a class of Heat Shock Proteins named by their molecular weight (HSP 70 is 70 kilodaltons). The molecules within the class of HSPs have different slightly different roles within a response to stress. Stress can compromise the structure of proteins and HSP70 promotes the survival of stressed cells by binding to partially-synthesized proteins and preventing them from aggregating and losing function. The binding of HSP70 can allow the proteins to re-fold, and regain function. Thus, when a species is exposed to stress, we should see an increase in HSP70 proteins. The function of HSP70 remains similar across a wide variety of species and is an indicator of general, non-specific stress.
October 11, 2010
Ran a PCR using the cDNA samples from 10/8/2010. The genes used in the PCR were MM_HSP70 and MM_Efb. Plate layout can be seen here. PCR was run in the Freidman Lab using the CFX96 Real Time system.
Calculations:
12.5ul x.5.5 = 68.5ul ImmoMix
.5 x 5.5 = 2.75ul Primer F
.5 x 5.5 = 2.75ul Primer R
1ul x 5.5 = 5.5 ul Syto-13
9.5 x 5.5 = 52.25ul H2O
Add 24ul of this to each PCR well and then add 1ul of template.
October 8, 2010
Used the cleaned up RNA samples, HC FL1 QPX and HC FL2 C to make cDNA using the Promega M-MLV Protocol. 1000ng of RNA was used in each reaction. Both samples were diluted by a factor of 10 in order to have a large enough volume to use in the reaction. After changing the the samples to cDNA, the goal is to investigate genes that previously did not work (see April 19, 2010) and see how they work with "usuable" DNA.
Calculations can be seen here.
August 19, 2010
qPCR from last time resulted in contaminated water samples and no amplification for the elongation factor primers. This suggests that there was likely some genomic DNA left over and which the cycling parameters were too limited to amplify.
Today, to make sure these are "working" primers, I isolated RNA from samples HC FL1 QPX M and HC FL2 C M. Concentrations for RNA samples:
http://genefish.fish.washington.edu/~srlab/Lexie/lexie%2008192010.bmp
RNA was cleaned using a precipitae. Concentrations can be seen below
http://genefish.fish.washington.edu/~srlab/Lexie/LEXIE%2008192010%20after%20precipitate.bmp
July 28, 2010
Prepared a qPCR using the cDNA from 10/23/10 and the Hard Clam Primers MM 18s_1 and Efb, both of which are normalizing genes. This qPCR should indicate whether the previous problems were from the primers or from the Hard Clam samples used. Plate layout and cycling parameters can be seen here
July 23, 2010
The qPCR from 7/16/10 again showed no positive results. Changed RNA to cDNA from Sam's RNA samples RNA HardClam Sperm and RNA HardClam Mix using the Promega M-MLV Protocol. 100ng of RNA was used in each reaction. Calculations showing how much RNA was used can be seen below. Both samples needed to be diluted, the sperm by a factor of 100 and the mix by a factor of 10.
HardClam Sperm:
Concentration= 1050ng/ul
Concentration after 1/100 dilution= 10.5ng/ul
100ng/10.5ng/ul = 9.5ul of RNA used
17.75ul- 9.5ul = 8.226ul of H2O used
HardClam Mix:
Concentration = 348ng/ul
Concentration after 1/10 dilution: 34.8ng/ul
100ng/34.8ng/ul = 2.87 ul of RNA
17.75- 2.87 = 14.87 ul H2O used
July 16, 2010
The qPCR from 7/14/2010 showed no positive results for the cDNA of any of the primers, though all water samples were clean. A new PCR using the same pool of cDNA and the MMGST-Pi and MM RACK-1 primers was created to attempt to get positive results. In addition the cycling parameters were changed. The plate layout and cycling parameters can be seen here.
July 14, 2010
Created a qPCR to test a cohort of primers for Mercenaria Mercenaria. The qPCR tested 8 different primers using the pool of cDNA from April 27, 2010 and water.
Calculations and Primers used can be seen here
Plate layout can be seen here.
July 8, 2010
PCR from 7/1/2010 showed poor amplification and poor efficiency along with two contaminated waters. In attempt to see if the poor efficiency is related to the primers or the cDNA, a new PCR was created using the pooled cDNA and MMGST Pi-1 primers from April 27, 2010. These results were positive, with an amplification for the cDNA around .4. The plate layout and cycling parameters for the new PCR can be seen here
July 1, 2010
The PCR results from 6/30/2010 showed very low efficiency (around 50%) and not very high amplification. To attempt to get better results an additional .25ul of Mg2+ was added to each PCR reaction. Instead of using the HSP70 gene, the 18s_1 gene was used, as a normalization gene, but other than that the PCR was identical and with the same cycling parameters. Plate layout can be seen here
Calculations for Mg2+ concentration:
5mmol(V1) = 1.5mmol (.025mL)
v1 = 7.4 E-4 mL or .075ul
June 30, 2010
Used the cDNA purified from 6/18/10 (CA32 and MA11) to run a PCR and see if the cDNA is now "usable". However, this time new cycling parameters (see below) were used., To normalize, the unpurified cDNA of the CA32 and MA11 samples (from 6/3/10) were included. The primers used were HSP70 and Efb, two normalizing primers. The volume of each PCR sample is 25uL. Plate layout can be seen here
Cycling parameters:
1)95C for 30 sec
2)60C for 1 min
For 50 cycles
June 18, 2010
PCR from 6/4/2010 showed poor results, so in order to make the cDNA usable, it needed to be purified. The RNA from CA32 and MA11 was purified by increasing the volume to 300ul and then purified using the Invitrogen Purification Kit. CA32 started with 11.18ul of RNA and was added to 288.882ul of water, while MA11 started with 14.128ul of RNA and was added to 285.872ul of water. After purified the resulting RNA was measured for absorbance, and concentration can be seen below
Concentrations are significantly lower, because the RNA was diluted by increasing the volume to 300ul.
The purified RNA was made into cDNA using the Promega M-MLV Protocol. 100ng of RNA was desired, so 3.471ul of CA32 RNA and 3.984ul of MA11 RNA was used (calculations can be seen below). Water was added to each sample to bring it up to 17.75ul and the protocol was run for 1ug of RNA
CA32: 100ng / 28.81ng/ul = 3.471ul cDNA
17.75ul - 3.471ul cDNA = 14.279 ul water
MA11: 100ng / 25.11ng/ul = 3.9824ul cDNA
17.75ul - 3.9824ul cDNA = 13.7675 ul water
June 4, 2010
Ran a PCR on the cDNA using primers 18s_1 and Efb. I chose to run the cDNA with these primers because they are control primers and do not change in response to stress. The plate layout can be seen here
June 3, 2010
DNased CA #32 and MA #11 and concentrations are seen below
Changed the DNased RNA from above into cDNA using the Promega M-MLV Protocol
100ng of RNA was used in the reaction. Using the concentrations above for CA32, 3.882ul of RNA was added to 13.868 ul of water to reach 17.75ul sample. For MA11, 0.872ul of RNA was added to 16.878ul of water to reach the 17.75ul sample. The protocol was then followed for 1ug of RNA.
May 27, 2010
Finished isolations for the Mercenaria mercenaria NRAC QPX3 by completing isolations on MAX #21, #22, #23, #24 and #25. Concentrations can be seen below
May 25, 2010
Isolated RNA from NRAC QPX3 samples #11, #12, #13, #14 and #15. Concentrations can be seen below.
May 21, 2010
Isolated RNA from NRAC QPX3 MAX samples #1, #2, #3, #4 and #5. Concentrations can be seen below
May 19, 2010
Isolated RNA from the remaining NRAC QPX3 MA samples, which includes #14, #15, #21, #22, #23, #24 and #25. Concentrations can be seen below.
May 13, 2010
Isolated RNA extractions on NRAC QPX3 MA#1, #2, #3, #4, #5, #11, #12 and #13. Concentrations are shown below.
May 12, 2010
Completed RNA extractions on NRAC QPX3 CA#25, #31, #32, #33 and #36. This concludes the CA samples from NRAC QPX3. Concentrations are shown below.
May 7, 2010
Completed RNA isolations on NRAC QPX3 CA #11, #12, #21, #22, #23, and #24. Concentrations are shown below.
May 6, 2010
Completed RNA isolations on NRAC QPX3 CA#1, #2, #3, and #11. Concentrations from each sample and the spectophotometer are shown below.
April 30, 2010
PCR from 4/19/2010 had clean water samples and positive amplification for cDNA. However, there was no clear pattern in QPX vs. control samples. In addition, samples 5 and 6 (both QPX) had very low efficiencies. This is likely due to something inhibiting the PCR and a possible explanation could be residue carry over from when the RNA was DNAsed. This reaction will be repeated to see if the same results can be replicated.
April 29, 2010
PCR from 4/27 came up with positive results for the cDNA and no contamination for all the water samples. Another PCR was prepared today with four water samples as a negative control, the 6 cDNA samples from before, and the pool of cDNA as a positive control. Plate layout can be seen here
April 27, 2010
Designed a primer for one the gene GST_Pi-1 listed below and prepared a PCR for it. Plate layouts can be seen here
April 19, 2010
Researched on NCBI.com other possible genes to create primers around for the hard clam samples. Found sequencing for 5 genes specific to the hard clam genome that are likely to be affected by exposing the organism to QPX.
Stress-Induced Protein STI- accession # GR209325
Toll-Like Receptor TLR- accession # GR209327
Tumor Necrosis Factor Receptor- accession # GR209326
C1q- TNF related protein- accession # GR209324
Beta-tubulin production, Halocynthia roretzi- accession # GO915225
April 16, 2010
Extracted DNA from a sea urchin tube foot and adult gigas gil using Quigen's DNEasy blood and tissue kit and protocol. Results from the spectophotometer are shown below. The sea urchin has a very low absorbance ratio and the 260/230 ratio is likely thrown off by the low quantity of the DNA.
April 14, 2010
Results from the previous qPCR showed clean water samples and an optimum at a Mg concentration at 1.5 and an optimum annealing temperature at 61.1C. A new qPCR was made using this Mg concentration and annealing temperature and including all 6 cDNA samples from the hard clam samples. Plate layout can be seen here
April 9, 2010
Results from previous qPCR turned out good. However, the efficiency for the Clam SPI gene was very low (ranged from about 25% to 80%) so we decided to optimize it using annealing temperature and Mg concentration. The plate layout can be seen here. The Mg concentrations ran from 1.5, which is included in the immomix, to 3. The temperature gradient went from 50 to 65C.
April 8, 2010
Results from the qPCR showed contaminated water samples in the NJC_48 primer series. The Clam-Prost primers had to significant flourescents so it is likely because the annealing temperature was too low. The Clam-SPI results had clean water samples and good results showing no significant differences between the QPX clams and the control clams. I prepared new samples using the NJC primers and the Clam-Prost primers in attempt to get the water samples clean. The annealing temperature was kept at 55C for the NJC primers but dropped to 50C for the Clam-Prost primers. Plate layout can be seen here
April 7, 2010
Ran a qPCR with the primers NJC 48, Clam-Prost Rec and Clam-SPI. These were the primers that were proved to have worked for this species based on the PCR from 2/24/2010. Plate layout can be seen here
April 2, 2010
Ran the PCR samples from yesterday out on a gel. All of them showed contamination, but the samples using the "normal" pipettes and tips showed a band that was significantly shorter than the others.
Lane 1: 100 base pair ladder
Lane 2-5: Lexie's samples using Sam's tips/pipettes
Lane 6-8: Sam's samples using Sam's tips/pipettes
Lane 9-12: Lexie's samples using normal tips/pipettes
April 1, 2010
Today Sam and I prepared samples in tandem with the same Taq MM, same water, same pipettes and same tips. The samples prepared were only water samples and never came in contact with cDNA. A fresh aliquot of Taq MM and water were used and they as well never came in contact with the cDNA. PCR was ran on samples with annealing temperature at 55C. These samples were made using Sam's pipettes and tips. A separate PCR was run using samples prepared from the same Taq MM and water, but using the standard pipettes/tips that I have been using in all the previous PCRs. This should allow us to isolate the source of the contamination, whether it be my process of making the samples, the pipettes themselves, or the Taq MM and water.
March 31, 2010
Ran the four water samples from 3/16 on a 1% gel with ethidium bromide. Samples were never in contact with cDNA but were contaminated. My only explanation for this would be that the Taq MM and/or PCR water is contaminated. From here, I think I will repeat the samples with ONLY water samples from both Sam and my taq and see if there are any differences from before.
March 16, 2010
Samples from 3/11/10 were run on a gel and were contaminated, again. This shows that because both mine and Sam's water samples came out contaminated, the contamination must be in the procedure. Four samples were then prepared with only water samples. The cDNA was never in contact with the prepared samples.
March 11, 2010
Sam ran a PCR using the Mercenaria 18s primers I used in the past reactions and a new stock. The results (seen here) show no water contamination, which means it is not the primers that were contaminated. New samples were prepared and run on a PCR using Sam's water and Taq MM vs. my water and Taq MM in attempt to identify where the contamination is coming from. The same primers and pool of cDNA was used.
March 10, 2010
New Mercenaria 18s samples were run on a 1% gel with ethidium bromide and results show contamination in the water samples, again. Samples will be run by Sam to see if the contamination is in the primer itself
March 8, 2010
Ran samples of Mercenaria 18s primer on a 1% gel with ethidium bromide. The first four samples are water and the last is cDNA. The results, seen below, show contamination of all water samples. A possible explanation of this could be contamination of the entire water sample. Samples were prepared for pcr, once again, but new pcr water was used.
March 4, 2010
Ran samples on a 1% gel with ethidium bromide.
Primer Mercenaria 18s looks like all the water samples were contaminated so samples were prepared for PCR again today with an annealing temperature of 55C. FLM 8b TIG 3 does not have bands at this annealing temperature so on a later date they will be re-run with annealing temperature at 50C. Clam-Prost REC looks successful as well as the NJC 48, with the exception of one water contamination.
February 24, 2010
Ran samples on a 1% gel. The first well in every row is a 100 base pair ladder. First row contained samples A1- A10, B1-B9. Row two contained samples B10, C1-C10, D1-D8. Last row contained samples D9, D10, E1-E10, F1-F10, all in that order. The first two rows contained 25ul of sample, the last row contained 15ul.
Results:
February 23, 2010
Samples were prepared for PCR. There were 6 different primers used to test which primers are effective. The primers used (in order) were Mercenaria 18s, Clam SPI, FLM 8a, FLM 8b TIG3, NJC 48, Clam-Prost- Rec 5. The template consisted of a pool of 7ul of each cDNA sample from 2/22/10 to test the efficacy of the primers.
MasterMix:
2 taq MM: 12.5ul x 11 = 137.5 ul
Primer-froward .5ul x 11 = 5.5 ul
Primer- reverse .5ul x 11 = 5.5 ul
Water 10.5 ul x 11 = 115.5 ul
6 separate mastermixes were created (one for each primer).
Sample assignments can be seen here.
February 22, 2010
Used samples from previous days to create cDNA. The concentrations were too low to make 1000ng so the following calculations were done to find the amount of RNA to use
1) 679ng/ 59.75ng/ul = 11.364ul
2) 679ng/ 38.26 ng/ul = 17.746ul
3) 679ng/67.94 ng/ul = 9.994 ul
4) 679ng/61.76 ng/ul = 10.994 ul
5) 679ng/ 64.42 ng/ul = 10.540
6) 679ng/ 53.93 ng/ul = 12.590
.679 ug x .5ul/1ug = .3395 ul of Oligo dT needed
The find the water needed for each reaction, the total RNA plus the oligo dT was subtracted from 18.25ul. The remaining volume of water was added.
MasterMix Composition
5ul 5x buffer x 6.6 = 33ul
1.25ul 10mM dNTPx 6.6 = 8.25 ul
.3395 ul of M-MLV enzyme x 6.6 = 2.2407
Samples are in chronological order in PCR plate starting under the 8.
February 19, 2010
Samples were prepared for PCR. Both DNased RNA and RNA was included to measure efficacy of primer as well as 4 water samples to bring total to 16. Samples assignments can be seen here.
Mastermix composition:
2x Taq mm 12.5 x 17.6 = 220ul
EukA 10mm .5 x 17.6 = 8.8 ul
EukB 10mm .5 x 17.6 = 8.8 ul
H2O 10.5 x 17.6 = 184.8
PCR was run with annealing temperature at 50C and extension time (72C) for 2:00.
DNased RNA results from spectophotometer
First two samples were not labeled but were
FL2, QPX and FL1, QPX
February 17, 2010
Results from spectophotometer from last 6 samples
Samples were numbered as follows
Sample 1:HC, FL2, QPX, G
Sample 2:HC, FL1, QPX, G
Sample3:HC, FL2, C, G
Sample 4:HC, FL1, C, G
Sample 5:HC BXI, C, G
Sample 6:HC, BXI, QPX, G
DNased the Gill samples from 2/16/2010
Calculations:
volume of sample used
1) 5ug/1.0469ul/ml = 4.776ul
2) 5ug/ 2.555ug/ml = 1/9569 ul
3) 5ug/.402ug/ml = 12.437 ul
4) 5ug/1.088ug/ml = 4.5956 ul
5) 5ug/1.7334ug/ml = 2.8845 ul
6) 5ug/ .78655ug/ml = 6.35687ul
February 16, 2010
Isolated RNA using common procedure for HC, FL2, QPX, G: HC, FL1, QPX, G: HC, FL2, C, G: HC, FL1, C, G: HC BXI, C, G: and HC, BXI, QPX, G
February 11, 2010
Ran samples on a 1% gel with ethidium bromide. Results can be seen here.
The sample tubes were placed numerically from left to right with a 100 base pair ladder on both ends. (See February 10, 2010 for sample names). The only sample that showed a band was the cDNA which indicates clean RNA and water samples.
February 10, 2010
Prepared samples for PCR using the DNased RNA.
Composition of mastermix:
2x taq mm = 96.35 ul
Primer-forward= 3.85 ul
Primer-reverse= 3.85 ul
H2O = 80.85 ul
Sample tubes 1-4 contained 24ul of mastermix and 1ul of water, used as a control
Sample tubes 5-7 contain 24ul of mastermix and 1ul of template (RNA or DNA)
Sample 5= DNased RNA sample 1 from 2/9/10
Sample 6= DNased RNA sample 2 from 2/9/10
Sample 7= cDNA sample 1 from 1/27/10
primer used= gigas_18s_f and gigas_18s_r
PCR:
Incubated with blocks
1) 95C for 10 min
2) 95C for 15 sec
3)55C for 15 sec
4) 72C for 30 sec
5) 72C for 10 min
2-4 cycled 40 times
February 9, 2010
Ran the Turbo DNase protocol on the two previous samples of RNA to try and remove any genomic DNA from the samples. The concentrations of the two samples were at 1093.25 ng/ul and 873.48 ng/ul. The calculations below show how much of each sample was used to get the entire sample to 5ug.
1) 1093.25ng/ul x 1ug/1000ng = 1.09325 ug/ul
5ug/1.09325 ug/ml = 4.5735 ul of Sample 1 used
50ul-4.573= 45.44 ul of H2O used in sample 1
2) 873.48 ng/ul / 1000 = .87348 ug/ul
5ug / .87348 ug/ul = 5.72428 ul of Sample 2 used
50ul - 5.724 ul = 44.276 ul of H2O used in sample 2
1 ul of buffer was added and the sample was incubated at 37C for 20 minutes. An addition 1 ul of buffer was added and then incubated again at 37C for 30 minutes.
Results of the spectophotometer are shown here
February 5, 2010
Ran out PCR on 1% gel with same procedure as 1/29/2010. Results can be seen here.
The samples from left to right are ladder, water, water, water, water, RNA1, RNA2, cDNA1, cDNA2, ladder. The results show that all four water samples were clean, which was the desired result. However, the cDNA and RNA both ran the same distance which indicates contamination of RNA with genomic DNA.
February 3, 2010
Ran PCR with same samples to try and get clean water samples. Same samples, procedure and cycles were used as January 28
January 29, 2010
Ran PCR on 1% gel with Ethidium Bromide. See results here
Results show 2 clean water samples and 2 which have bands on them. cDNA and RNA are both amplified which indicates a contamination of the RNA with genomic DNA.
January 28, 2010
Prepared samples for PCR.
Sample tubes 1-4 contain 24ul of mastermix (see here for composition) and 1ul of water and are to be used as control group
Sample tubes 5-8 contain 24ul of mastermix and 1ul of template (RNA or DNA)
Sample 5= RNA sample 1 from 1/25/10
Sample 6= RNA sample 2 from 1/25/10
Sample 7= cDNA sample 1 from 1/27/10
Sample 8= cDNA sample 2 from 1/27/10
primer used= gigas_18s_f and gigas_18s_r
PCR:
Incubated with blocks
1) 95C for 10 min
2) 95C for 15 sec
3)55C for 15 sec
4) 72C for 30 sec
5) 72C for 10 min
2-4 cycled 40 times
January 27, 2010
Reverse Transcription with RNA extracted from 1/25/2010 using only samples 1 and 2
Sample 1- 1093.25 ng/ul
Sample 2- 873.48 ng/ul
Amount of RNA used (Numbers indicated sample number)-
1) 1000ng/1093.25ng/ul = .9147 ul RNA
2) 1000ng/ 873.48ng/ul = 1.11448 ul RNA
Water added to RNA to get a total solution of 17.75 ml
1) 17.75ul- .9147ul = 16.84 ul of Water added
2) 17.75ul- 1.1448ul= 16.61 ul of Water added
Heated samples to 70C for five minutes in the thermocycler
Need .25ug per every .5ul of oligo dT, so .5ug was used
Mixed together Buffer, dNPT and M-MLV RT for both solutions to minimize error, added 10% to make sure it is not limiting
5ul x 2.2 = 11ul of Buffer
1.25ul x 2.2 = 2.75ul of dNTPs
.5ul x 2.2 - 1.1ul of M-MLV RT
Incubated at 42C for 1hr in thermalcycler.
Heat inactivate at 95C for 3 min.
Then cooled down to 4C
Stored at -20C
January 25, 2010
RNA extraction with standard protocol, results are as follows.
Species are Willapa Bay 51 x 35 Dec 09 RO26
Tissue used is gill tissue
| Sample Number |
Length |
mg of tissue Used |
| 1 |
49.5mm |
90mg |
| 2 |
51.5mm |
80mg |
| 3 |
45mm |
110mg |
| 4 |
49mm |
60mg |
January 21, 2010
Introduction to content and safety precautions
Learned how to manage the e-lab notebook and calender