Today I ran a gel on the 4 samples, using the same methods as on Tuesday July 20, 2010 (see below), except I used a 12-toothed comb. Results can be seen here: http://genefish.fish.washington.edu/~srlab/Christina/DSCF0829.JPG This work marks the end of my internship. Though no conclusions were made about DNA methylation, certain questions were brought up about the published sequences of C. gigas hsc70 and hsp70 genes. To understand fully what the sequence of the samples we have in house, we'll have to ask further questions. When we understand better what the sequences are we can utilize the primer set that I had ordered. I ran this last gel to further that goal. The size of the sequence should be approximately 118 bp if the primers are really isolating the part of the gene we think they are. Unfortunately the only ladder we have is the HyperLadder I. The lowest notch on that scale is 200. The samples do appear to be under 200 bp. That's a start.
Friday August 6, 2010
The results from last weeks qPCR are shown here: http://genefish.fish.washington.edu/%7Esrlab/Christina/20100730qPCRcm.bmp
Again all samples resulted in amplification. Are there no CCGG sites? To verify what these primers are actually isolating I will run PCR on them as I did with the other set of primers that Mac designed. Ran exactly as found in July 19th's entry, except for the fact that the forward and reverse primers are separate so volumes are halved. Ran the same cycling parameters found under Apex in the menu. Will run a gel next week.
Friday July 30, 2010
Ran real-time PCR on the new set of primers. This set of primers was designed to include the CCGG combination near the terminal end of the AJ318882 (and theoretically the AJ305315, thought they may be the same gene). These primers were designed and ordered on Thursday July 15 (see below). The concentrations of the master mix can be found here: https://spreadsheets.google.com/ccc?key=tHWBE7R2cwlYE_-K1-LhM9Q&authkey=CLucjY8M#gid=0. The PCR was run on the Opticon. The cycling profile chosen was SYBR cDNA 55melt 2reads, and the file was saved as 20100730_110427 in the data folder. The layout can be found here: http://genefish.fish.washington.edu/~srlab/Christina/20100730%20PCR%20layout.jpg The H U and M wells are the positive controls.
Thursday July 29, 2010
Reviewed paper and sequences in Geneious. PCR resulted in only one product which refutes the theory provided by below paper, which states that hsc70 and hsp70 are two different genes.
Tuesday July 20, 2010
Ran an agarose gel for the 6 samples PCR'd yesterday- (AS1, ANS1, CS1, CNS1, 2 blanks). Used 1.5% solution Agarose (0.75g:50mL). Added 4uL Ethidium Bromide. Used 8 teeth comb with teeth size, 1 mL, and the Hyper Ladder1 to measure. From left to right: Ladder/AS1/ANS1/Control/CS1/CNS1/Control/Empty. Picture of hyperladder key here: http://2008.igem.org/wiki/images/a/a2/BCCS-080812-HyperLadderI_for_electrophoresis.PNG. Picture of gel results here: http://genefish.fish.washington.edu/~srlab/Christina/20100720%20PCR%20C.giga%20gel_cm.jpg
note: cannot view gel. try embedding it into your notebook-
Monday July 19,2010
hsc70 and hsp70 in C. gigas, are two separate genes,with the same coding dna but hsc70 includes introns. This is guessed to be for rapid translation in times of need. To see more read this paper http://www.ncbi.nlm.nih.gov/pmc/articles/PMC514856/. To see what size I'm working with, I ran normal PCR with Apex 2xRxn. Recipe for master mix found here: https://spreadsheets.google.com/ccc?key=tbYkXbkcdsq7_dcfPnKYY0Q#gid=0. Cycle designed to run 95C for 10 min. Then 40x (95C for 30sec, 55C for 30 sec, 72C for 2 min) and a final 72C for 10 min. This cycle is saved under Main>Apex>Block. Will run a gel tomorrow AM.
Friday July 16, 2010
qPCR results look questionable. All enzymatic treatments produced results. All DNA samples were clean, so it was not a contamination error. There may be a concern about non-coding DNA involvement since it is not included in the gene sequence we are using (AJ138882) in Geneious (to build primers and analyze CCGG, CpG Islands, etc). I then compared AJ138882 to other accession numbers in the NCBI database. I compared 4 accession numbers, 1 for cds of hsp70, 2 for genomic hsp70 and one genomic for hsc70 in C. gigas.The closest match was with hsc70 and it included introns and exons. It also matched the other cds's well. The comparisons went as follows:
1. AJ318882=(hsp70 cds that we've designed primers and such off of)
2. FJ707369=(a much shorter allele with genomic info)
3. AJ305315=(hsc70)
4. AB122063=(mRNA includes introns)
1vs2=ok match
1vs3=ok match
1vs4=poor match
2vs3=ok/poor match
2vs4=poor match
3vs4=poor match
Thursday July 15, 2010
Today I got a brief intro to Geneious. The C. gigas// HSP70 cds is on there with the accession number AJ318882. Mac has already designed and received primers containing a CCGG site in the CpG island of this gene. I designed and ordered a new set of F/R primers isolating the second CCGG site in the latter part of the cds. They're about 20bp in size.
Ran qPCR with the primers that Mac designed. Layout of plate can be found at this link https://docs.google.com/leaf?id=0B_GhJMWO6l8qYTg4ZWQxYzAtMDM2NC00NjUzLWI3MWYtMmU4Y2RlZGM1NjUw&hl=en&authkey=CKrL7ewM. The volumes used in the qPCR master mix can be found at this link https://spreadsheets.google.com/ccc?key=tF-FzZy0Q8My1v7woo-2zQg&hl=en#gid=0.
Friday July 9, 2010
Performed restricted enzyme digestion for four DNA samples. Samples chosen: (one from each treatment) CS1, CNS1, AS1, ANS1. All samples diluted to 25ng/uL, then added to enzyme/buffer soln and stored at 37C for 4 hours. Each of the four DNA samples were treated with
1. HpaII enzyme with Buffer 1
2. MspI enzyme with Buffer 4
3. Water
For a total of 12 tests. Dilutions and master stocks found at this link: http://spreadsheets2.google.com/ccc?key=taK8DNvuGVBNA4hnreM3fMQ&hl=en#gid=0
All samples heat deactivated after four hours and stored in the -20C freezer. PCR to be run next week.
Thursday July 8, 2010
Today is the first day of a one credit internship. I will be in ~6hrs/week for five weeks. My initial project will be aiding in the stress-induced DNA methylation investigation. I started today by running specs on all the original DNA samples of the CO2/Mechanical stress experiment.
