Results from Western? -
12/2/10
Today in lab I normalized all my protein to 1700ug/ml. I then ran a SDS-PAGE gel with 5 replicates. After the gel was run, I started the western blot procedures. I ran the procedures through incubating the membrane in the Primary antibody solution, which I let incubate over night so I can finish the rest of the steps on 12/3.
11/30/10
Today in lab I extracted protein from the gill tissue of the big beef creek oysters. Before I had a chance to do that, the gill tissue was split in half, so that half could be used for RNA extraction. After extraction, I quantified the amount of protein extracted so that I can normalize all the protein samples. On Thursday, I will normalize the samples, run a SDS-PAGE gel and a western blot.
11/16/10
Oyster Project Notes
Copper was added to the treatments in lab on 11/16/10. 1.3 ppm was added. This came to 6.5 ml per 5 L (amount in buckets). Today in lab, all the tubes for oyster tissue were labeled in preparation for Friday. On 11/18/10, the Vibrio will be added to the specified treatments and termination will occur on 11/19/10. After termination, gill, mantle and hemolymph tissue will be harvested and frozen for work next week in lab.
11/14/10
Oyster Project Notes
No Lab work was done in lab on 11/9, however, project logistics were discussed.
Oyster Collection will occur on Thursday 11/11 from two sites: Big Beef Creek (pristine), and Lynch Cove (urban). These two groups will be treated with vibrio and/or copper for response. Oysters will be acclimated over the weekend in buckets held in 12˚C water bath. 64 oysters will be taken, 32 from each site. A control, copper, vibrio, and copper+vibrio treatments will be run each with 2 replicates of 4 from each site. Copper will be added to treatements 11/16/2010, then Vibrio will be added 11/18/2010, Oyster samples taken (mantle, gill, hemolymph) 11/19/2010. Tests will be run on the samples including protein, RNA, DNA extractions and quantified via pPCR, dotblot. I have chosen to run protein tests on the oysters.
11/3/10
Summary
The purpose of this lab was to perform qPCR to measure the HSP-90 gene expression as well as to measure cytosine methylation using dot blot and chromogenic immunedetection methods.
Materials and Methods
Chromogenic Immunodetection
20 ml of blocking solution was prepared using 14 ml ultra filtered water, 4 ml of blocker/diluents part A and 2 ml of blocker/diluents part B. the membrane was placed in 10 ml of blocking solution in a covered plastic dish. This was incubated for 30 minutes on a rotary shaker set at one rotation per second. The blocking solution was decanted and the membrate was rinsed with 20 ml of water for 5 minutes then decanted again. 10 ml of primary antibody solution was made using 10 ml blocking solution and 2 ul 5-MeC antibody. The membrane was then incubated with the primary antibody solution for 1 hour. The primary antibody wash was decanted and the membrane was washed for 5 min with 20 ml of TBS-The membrane was then incubated in 10 ml of secondary antibody solution for 30 minutes then decanted. The membrane was washed for 5 minutes with 20 ml of TBS-T, decanted and rinsed three more times. The membrane was rinsed with 20 ml of water for 2 minutes, decanted and repeated twice. The membrane was then incubated in 5 ml of Chromogenic Substrate until color began to develop. The membrane was then rinsed with 20 ml of water for 2 minutes, decanted and repeated twice. The membrane was then dried with a clean piece of filter paper.
qPCR Procedure
The HSP-90 primer designed as part of a previous lab was re-hydrated to 100 mM as a stock solution and then 100 ml of 10 mM working stock was made for use in qPCR. After the primer was re-hydrated, the master mix was prepared using a ratio of 25 ul Master Mix, 2x(immomix), 2 ul syto-13 dye, 2.5 ul upstream primer, 2.5 ul downstream primer, and 16 ul of ultra pure water. The 48 ul of the master mix was added to six wells of a white PCR plate. 2 ul of the cDNA, RNA and water (as a negative control) were added to their respective wells. The wells were then capped and spun before the PCR was run.
Results
http://img.skitch.com/20101103-t4srwywxn6imd8w17jm9gcrh94.png
Conclusion
The results from the qPCR are not what I expected. I expected to see two nice lines from the cDNA samples, which was seen, however, I expected to see no amplification from the RNA, which was not the case. This suggests that my RNA samples were contaminated with carryover DNA when it was originally processed. To take care of this in future labs I would need to be more careful in my RNA extraction. Also, I could put an enzyme in with my RNA that breaks down DNA and this problem would be taken care of. I think the RNA carryover came from the lab where we were extracting the RNA and we were only supposed to take one of the layers. I think I may have taken the wrong one as my RNA yield as seen from previous labs was really low and I had some carryover DNA.
The dots on my dot blot did not show up which suggests the test failed.
Reflection
The purpose of the qPRC this week was to look at the gene expression of the genes we selected for with our primers. This could be used in any lab setting where there is a gene of interest. I am not sure what the purpose of the cytosine methylation dot blot was and I would like to get more information about that.
10/29/10
Summary
The purpose of this lab was to check the success of our PCR by running the product in agarose gel as well as to measure cytosine methylation using dot blot and chromogenic immunedetection methods.
Materials and Methods
Electrophoresis Procedure
The gel was placed in the gel box and filled with 1x TAE buffer solution
The combs were removed from the wells. 5 ul of 100bp ladder was loaded into the far left lane. 25 ul of the PCR sample was loaded into the gel and the gel was run at 100 V for 60 minutes, 150 V for 15 minutes and 85 V for 20 minutes. The gel was then visualized under the UV transilluminator.
Methylated Cytosine Dot Blot Procedure
The DNA sample used in the following procedures was from Drosapilla. First, five snap cap tubes were labeled and appropriate concentrations were made (0.8, 0.4, 0.2, 0.1, and 0.05 ng/ul). The DNA was set aside to prepare the dot blot vacuum manifold. The nylon membrane was cut to fit 72 wells of the manifold and soaked in 6x SSC for 10 min. filter paper was cut to the size of the nylon and wet in 6x SSC. The manifold was assembled with the membrane lying on top of the filter paper. The DNA was denatured in boiling water for 10 minutes and immediately transferred to ice. The vacuum was then switched on and 500 ul of 6x SSC was placed in each well and allowed to filter through. The DNA was spun down for 5 seconds. The entire contents of the DNA was then transferred to its respective well. The samples were then allowed to filter through. While the samples were being filtered through, filter paper cut to size was soaked in denatureation buffer. Once filtered though, the manifold was dismantled and the membrane was transferred to filter paper soaked in denaturation buffer and allowed to sit for 10 minutes. The filter paper was then soaked for 5 minutes in neutralization buffer. The membranes were then placed on dry filter paper and allowed to dry. The dryed blot was wraped in plastic wrap and placed DNA side down onto the UV transluminator for 2 minutes.
Chromogenic Immunodetection
20 ml of blocking solution was prepared using 14 ml ultra filtered water, 4 ml of blocker/diluents part A and 2 ml of blocker/diluents part B. the membrane was placed in 10 ml of blocking solution in a covered plastic dish. This was incubated for 30 minutes on a rotary shaker set at one rotation per second. The blocking solution was decanted and the membrate was rinsed with 20 ml of water for 5 minutes then decanted again. 10 ml of primary antibody solution was made using 10 ml blocking solution and 2 ul 5-MeC antibody. The membrane was then incubated with the primary antibody solution for 1 hour. The primary antibody wash was decanted and the membrane was washed for 5 min with 20 ml of TBS-The membrane was then incubated in 10 ml of secondary antibody solution for 30 minutes then decanted. The membrane was washed for 5 minutes with 20 ml of TBS-T, decanted and rinsed three more times. The membrane was rinsed with 20 ml of water for 2 minutes, decanted and repeated twice. The membrane was then incubated in 5 ml of Chromogenic Substrate until color began to develop. The membrane was then rinsed with 20 ml of water for 2 minutes, decanted and repeated twice. The membrane was then dried with a clean piece of filter paper.
Results and Conclusion
Four distinct bands, identical in each of the experimental wells, were seen under the 1000 bp level. The most distinct band was seen between 800-600bp. The two control wells containing water reveiled two faded bands which could be a result of “primer dimmers”. These results suggest that even though I had a low yield of RNA in previous labs, my PRC was successful yielding a high amount of cDNA. I could not load the picture of the gel into the lab. Dot blot was not completed by the end of lab, so results from the procedure are yet to be determined.
Reflection
The purpose of this week’s lab was to run check to success of our PCR from lab three. These procedures could be used in any experiment using cDNA to make sure that the PCR was done correctly and yielded enough cDNA. I am not sure what the purpose of the cytosine methylation dot blot was and I would like to get more information about that.
10/20/10
Summary
The purpose of this lab was to change our extracted RNA into cDNA using reverse transcription. After that the gene of interest (P450) was amplified using PCR.
Materials and Methods
Reverse Transcription Protocol
The stock RNA from previous labs was taken out of the freezer and mixed by inverted several times. A 0.5 ml PCR tube was labeled with my initials and 5 ul of stock RNA, 1 ul of oligo dT, and 4 ul of nuclease free H2O were added. The mixture was incubated for five minutes at 70 C in a thermocycler then transferred immediately to ice. After cooling, 5 ul M-Mlv 5x reaction buffer, 5 ul dNTPs, 1 ul M-MLV RT and 4 ul nuclease free H20 were added. The mixture was incubated for 60 minutes at 42 C and then heat inactivated at 70 C for 3 minutes. The sample was then spun down in a centrifuge and transferred to ice.
PCR Protocol
Four 50 ul reactions were prepared, two containing cDNA and two a negative control. In a 1.5 ml tube, a reaction master was made containing, 250 ul GoTaq Green Master Mix, 15 ul of forward primer, 15 ul reverse primer, and 108 ul nuclease free H2O. 48 ul of the master mix was added to each of the 4 0.5 ml PCR tubes. To two of the tubes, 2 ul cDNA was added and to the remaining two, 2 ul of nuclease free H2O was added. The tubes were spun to pool the liquid at the bottom of the tubes. The reactions were loaded into the thermocycler, where the reactions were run through a thermal cycling profile then stored at -20 C.
Agarose Gel Pouring Protocol
Two grams of agarose was mixed with 150 ml of 1x TAE in a 1 L flask. The solution was microwaved for 3 minutes. The solution was then cooled before 12 ul ethidium bromide was added. The mixture was mixed thoroughly by swirling, then poured into a gel tray. Gel combs were added, and once set, wrapped in plastic and placed in a fridge for the next week.
Results and Conclusion
All the steps run in lab were procedural and therefore there are no results to report.
Reflection
The purpose of this week’s lab was to run PCR on our RNA samples so that the results can be viewed next week in lab. The procedures run lead up to the PCR and the purpose of making the gel was to use next week in sorting out the results of our PCR.
10/15/10
Summary
Today in lab I ran SDS-PAGE on isolated protein from pacific oyster gill tissue (C. gigas). I also finished extracting RNA from gill tissue of the same animal. The extracted RNA was then quantified using a Nanodrop spectrophotometer.
Materials and Methods Nicely written -
SDS-PAGE Protocol
The protein quantification process began from thawing the protein extract form last week and inverting several times. 15 ul of the protein was added to a fresh 1.5 ml screw cap tube along with 15 ul of 2x reducing sample buffer. The sample was mixed by flicking and briefly centrifuged. The sample was boiled for five minutes. After boiling, the sample was immediately centrifuged for 1 minute. The entire sample was loaded into the appropriate well and the gel was run for 45 minutes. After 45 minutes, the gel was removed, stained, and rinsed with 10% acetic acid until the protein bands became visible.
RNA Extraction Protocol
The homogenized tissue sample from lab one was incubated at room temperature for 5 minutes to allow for thawing. Under a fume hood, 200 ul of chloroform was added to the sample. The sample was then vortexed for 30 seconds. After vortexing, the sample was allowed to incubate at room temperature for 5 more minutes. After incubating, the sample was spun in a refrigerated microfuge for 15 minutes at max speed. When finished spinning, the tube was gently removed to avoid mixing. The top aqueous phase was transferred to a fresh screw cap tube. 500 ul of isopropanol was added to the new tube. The contents was mixed by inverting until the solution appeared uniform and allowed to incubate at room temperature for 10 minutes. After incubating, the sample was spun for 8 minutes in a refrigerated microfuge. The supernatant was removed after spinning leaving a small pellet at the bottom of the tube. 1 ml of 75% EtOH was added then the contents was vortexed briefly to dislodge the pellet from the side of the tube. The sample was spun for five minutes. After spinning, the supernatant was removed taking extra care not to remove the pellet. The pellet was allowed to dry to remove all excess EtOH. The pellet was resuspended in 100 ul of 0.1% DEPC-H2O and mixed by pipetting. The tube was incubated at 55 C for 5 minutes to stabilize RNA then transferred to ice. The RNA yield was then quantitated using a Nanodrop spectrophotometer with the manufacturer’s procedures.
Results
SDS-PAGE
I could not upload the picture of the gel, however, the sampled showed three distinct bands about halfway down the gel. What lanes where your samples? -
RNA Quantification Results
Nanodrop absorbance at 260nm = 9.6
260/280 ratio = 1.73
Conclusions
The SDS-PAGE gel showed three strong bands of protein. The entire contents of my sample was not loaded into the well which might have decreased the strength of some of the weaker bands. The three strong bands were around the BSA, Alcohol Dehydrogenase and Carbonic Anhydrase bands when compared to the protein standard. If this gel were part of a larger project I would most like rerun the gel, however, this was a good learning experience.
The RNA extraction produced a low absorbance with a weak curve on the Nanodrop. The ratio also indicated there was some contamination of the samples. The absorbance is low, however, it seems that there might still be enough RNA in the sample to continue with the labs in the following weeks.
Reflection
The SDS-PAGE gel was fun to run and interesting to see. The purpose of the gel was to look at the densities molecular weight -
![Paul_Lab 2 [Compatibility Mode]](http://img.skitch.com/20101021-gqtsmdwpu57nfmwngtdhedj8aw.preview.jpg)
![Paul_Lab 2 [Compatibility Mode]](http://img.skitch.com/20101021-pawituwtsu41y3pw5q8isejmqs.preview.jpg)
Lab 1
Summary
Today in lab I isolated protein from pacific oyster gill tissue (C. gigas). I also started extracting RNA from gill tissue of the same animal. The concentration of total protein in the tissue was determined using the Bradford protein assay. TriReagent was added to a different tissue sample to begin the RNA extraction process.
Materials and Methods
The RNA extraction process began with adding 500 uL TriReagent to the tube containing the 24 mg piece of pacific oyster (C. gigas) copper treated gill tissue. The tissue was gently homogenized using a disposable pestle and occasionally vortexing. After homogenizing was completed, an additional 500 uL of TriReagent was added and the contents were vortexed for 15 seconds. The sample was then labeled “C. gigas 24mg Cu Treatment B” and placed in a -80°C freezer.
The protein extraction began with recording the weight of the oyster tissue which was 23 mg. 500 uL of CellLytic MT was added to the tube containing the tissue. The tissue was homogenized using a disposable pestle. After the sample was homogenized, the tube was closed and inverted several times. The sample was then spun at max speed for 10 minutes in a refrigerated microfuge. A new tube was labeled “Protien, C. gigas, gill, PP, 10/5” and then the supernatant from the microfuged tube was transferred to the new tube. A fresh tube was labeled “Protien BA, PP, 10/5”. The protein sample was diluted 1:2 by adding 15 uL of the protein and 15 uL of DI water. In a second tube labeled “blank”, 30 uL of DI water was added. To both tubes, 1.5 mL of Bradford Reagent was added. The tubes were inverted several times then incubated for ten minutes at room temperature. The ‘blank’ was mixed several times with pipetting and transferred to a 1000 uL disposable cuvette. The spectrophotometer was zeroed using the blank. The same procedure was used to transfer the ‘protein’ sample to a new disposable cuvette. The absorbance was then measured in the spectrophotometer. After the value was recorded, the sample was mixed via pipetting and measured again. The two values were then averaged. The protein concentration was then back calculated using the standard curve y=1013.9x. The protein sample was then stored at -20°C.
Nicely written. -
Results
Spectrophotometer readings: #1=0.101
#2=0.094
Average=0.0975
y= 1013.9(0.0975)=98.855 X 2(dilution)= 197.71 mg/mL
Conclusions
Due to the fact that I have only completed part one of RNA and protein extraction and analysis, there are not many results to discuss. The protein concentration was found to be 197.71 mg/mL. This number might be slightly low due to the fact that I had some problems homogenizing the tissue sample. The homogenization process took longer than normal for me and not all the tissue became completely homogenized. This might lead to a slightly lower protein density concentration -
Reflection
I think that the purpose of this lab was to begin RNA extraction so that once we get the RNA we can use it to make copies Yes! using PCR! -
NCBI Sequences
1. AY713403 “Crassostrea gigas protein kinase C receptor mRNA, partial cds”
2. AB234293 “Crassostrea gigas taut mRNA for taurine transporter, complete cds”
3. EF687776 “Crassostrea gigas heat shock protein 90 mRNA, complete cds”
Good sequence picks. These are all mRNA sequences so they can be used to design primers for the gene expression qPCR assays will be ding in class. I would just pick which of these genes interests the you most. -