Digest master mixes are here. Digests were incubated @ 37C for 2hrs. and then heat inactivated @ 80C for 20mins.
Tag Archives: gill
gDNA Isolation – Various gigas samples (continued from yesterday)
Pelleted residual tissue 10mins @ 10,000g @ RT. Transferred supe to new tubes. Precipitated DNA with 0.25mL 100% EtOH. Incubated 3mins @ RT. DNA was pelleted 5mins @ 5000g @ RT. Supe was removed, pellets were washed with 1mL 75% EtOH (x2). Supe was fully removed and the DNAs were resuspended in 300uL 8mM NaOH (made 7/9/10 SJW).
1M HEPES (provided with DNAzol) was added at a 1:100 dilution to achieve a pH = 8.0. This was based on the DNAzol protocol calculations (For 1mL of 8mM NaOH, use 101uL of 0.1M HEPES = pH 8.0).
Samples were spec’d on NanoDrop 1000. Used a sample with 8mM NaOH and 1M HEPES as a blank to match the pH = 8.0 of the samples.
Results:
260/280 ratios look good for all samples. Most of the samples have mediocre 260/230 ratios. Yields are excellent for all samples.
gDNA Isolation – Various gigas samples
Placed ~20mg fragments of tissue in 250uL DNAzol. Added 1.35uL of Proteinase K (Fermentas; 18.5mg/mL) to reach a final concentration of 100ug/mL. Incubated RT, O/N, end-over-end rotation. Will complete DNA isolation tomorrow.
Sample List:
Vt Gigas Live #3 Gill 24E (from 20080828; Tatyana’s notebook)
Gigas Control #2 Gill 24E (from 20080828; Tatyana’s notebook)
NB-1209-10 (RNA Later)
SB-1209-14 (RNA Later)
WB-1209-09 (RNA Later)
0629 gill 5aza
0629 gonad 5aza
0629 mantle 5aza
Samples Received – Hard Clam samples from Rutgers and MBL
*Important Note: These were received while I was out of lab. This notebook entry was added 20101021*
Received sets of gill tissue and hemolymph in RNA Later from Rutgers (Emily). Here’s the note that was included with the samples.
Received set of gill tissue in RNA Later MBL (Scott Lindell).
All samples were stored @ -80C.
gDNA Isolation – Mac gigas gill samples (continued from yesterday)
Continued with gDNA isolation from yesterday’s samples. Samples were gently pipetted up and down to further dissolve remaining tissue, although tissue did not dissolve entirely. Pelleted residual tissue 10mins @ 10,000g @ RT. Transferred supe to new tubes. Precipitated DNA with 0.25mL 100% EtOH. Incubated 3mins @ RT. DNA was pelleted 5mins @ 5000g @ RT. Supe was removed, pellets were washed with 1mL 75% EtOH (x2). Supe was fully removed and the pellets were resuspended in 200uL 8mM NaOH (made by Amanda Davis 5/20/10).
1M HEPES (provided with DNAzol) was added at a 1:100 dilution to achieve a pH = 8.0. This was based on the DNAzol protocol calculations (For 1mL of 8mM NaOH, use 101uL of 0.1M HEPES = pH 8.0).
Samples were spec’d on NanoDrop 1000 on 20100607. Used a sample with 8mM NaOH and 1M HEPES to match the pH = 8.0 of the samples.
Results:
Overall DNA quality looks good (based on 260/280 ratios). Yields seem satisfactory. Will run samples on gel to verify gDNA integrity (see below).
250ng of each sample was run on a 1.2% TAE agarose gel. Gel was run on 20100607.
The results are pretty interesting.
Most of the R51 samples are pretty good looking (i.e. high molecular weight band, little smearing), but there are some samples that show a high degree of degradation (e.g. #18, #19).
All of the R37 samples look STELLAR (i.e. high molecular weight band, no smearing)!
The stark differences between the R51 samples and the R37 are intriguing. Although not currently verified (as of 20100607), I suspect that the amount of tissue stored in RNA Later possibly contributes to the long term integrity of the DNA, as nearly all of the R37 samples had very little tissue in the RNA Later. Whereas the R51 tissue samples were significantly larger in virtually every sample. I will do a visual inspection of the tubes to see if there is indeed a correlation between tissue size and apparent DNA quality.
gDNA Isolation – Mac gigas gill samples
Set up gDNA isolation from the following samples:
R51 01 – 20 (sample #11 was processed yesterday)
R37 01 – 03, 06 – 13, 15, 16
Samples were thawed from -80C. Tissue was removed from RNA Later (RNA Later gDNA isolation protocol; this protocol doesn’t indicate that anything needs to be done to the sample prior to gDNA isolation) and ~25mg was cut from each and placed in 0.5mL of DNAzol. 2.7uL of Proteinase K (Fermentas; 18.5mg/mL) was added to each tube to reach a final concentration of 100ug/mL. The digests were incubated @ RT O/N.
Package – Hard Clam gill tissue/hemolymph in RNA later
Rec’d package from Rutgers (Emily Pearson) containing two large Ziplock bags on “wet” ice, each of those containing smaller bags with sample tubes in them. One large bag contains gill tissue samples and the other large bag contains hemolymph samples. Samples will temporarily be stored @ 4C until they can be catalogued and boxed by Lexie later today.
qPCRs – Tim’s Adult Gigas gill cDNA (from 20091009)
Duplicates of earlier qPCRs.
Primers: Cg_P450 and TNFRAF_5’/3′.
qPCR set up and plate layout are here.
Results:
Duplicates of earlier qPCRs.
Primers: Cg_IkB_F997, R1213 and Cg_Prx6_F270, R439.
qPCR set up and plate layout can be found here.
Results:
qPCRs – Tim’s Adult Gigas gill cDNA (from 20091009)
Duplicates of earlier qPCRs.
Primers: Cg_HIF1 and IL17 Iso D.
The previous version of IL17 primers used (IL17 internal) were NOT the ones used for the paper. The IL17 Iso D are the correct primers and are the same ones that Tim previously used on the juvenille gill samples.
qPCR set up and plate layout can be found here.
Results:
Duplicates of earlier qPCRs.
Primers: EF1 and IL17 Iso D.
The previous version of IL17 primers used (IL17 internal) were NOT the ones used for the paper. The IL17 Iso D are the correct primers and are the same ones that Tim previously used on the juvenille gill samples. qPCR set up and plate layout can be found here.
Results:
Hard Clam Challenge – QPX Strain S-1 (continued from yesterday)
All clams appeared to be alive and well. Most had their siphons out when I arrived to start collecting tissues. Clams were shucked after 24hr challenge. Gill and mantle samples were collected in separate 1.5mL snap cap tubes, stored briefly on ice and transferred to -80C in “Hard Clam QPX Challenge 12/2/2009.” box.