Received 82 gill samples in RNA Later in 3 microfuge tube racks from MBL (Scott Lindell). Samples were catalogued, boxed (1 box) and stored at -80C.
The live clams were received in two bags one containing ~12 labelled MA4 and one containing ~12 labelled BX1. Clams were NOT counted and the quantity may be different. Clams were temporarily stored @ 4C.
Rec’d package from Scott Lindell @ MBL containing 65 screw cap tubes in a white microtube rack. All tubes are tissues in RNA Later (presumably). One sample (MA4-5) was lost during a brief centrifugation to get tissue sample unstuck from top of tube and in to RNA Later solution. The head of the tube snapped off and the entire tube/sample was obliterated in the rotor. Also, it appears as though all the tubes leaked RNA Later solution during transport. Samples were temporarily stored @ 4C and will be catalogued/transferred to -80C.
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.
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.
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.
Continued with gDNA isolation from yesterday’s samples. Additionally, isolated gDNA from R51 01, but homogenized the tissue (using disposable 1.5mL mortar/pestle) in 0.5mL of DNAzol and topped off to 1.0mL. All 3 samples were gently pipetted up and down to further dissolve the tissue. For those samples that were subjected to Proteinase K digestion, I transferred 100uL of the solution to a new tube containing 1mL of DNAzol, as described in the DNAzol protocol (see “Notes, #5″ part of protocol). Tubes were incubated 10mins @ RT.
Pelleted residual tissue 10mins @ 10,000g @ 4C. Transferred supe to new tubes. Precipitated DNA with 0.5mL 100% EtOH. Incubated 3mins @ RT. DNA was pelleted 5mins @ 5000g @ 4C. Supe was removed, pellets were washed with 1mL 75% EtOH (x2). Supe was fully removed and the pellets were resuspended in 8mM NaOH (made by Amanda Davis 5/20/10). See below for volumes:
R51 11 – 50uL
R51 01 – 100uL
R51 01 homogenized – 200uL
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).
Samples were spec’d on NanoDrop 1000. Used a sample with 8mM NaOH and 1M HEPES to match the pH = 8.0 of the samples.
Results:
The 260/280 ratios for all samples are great. Yields are significantly lower than I was expecting. However, for R51 11 and R51 01, only 100uL (1/5th) of the digestion was used as described in the protocol. I may recover the remainder of the R51 11 gDNA since remaining tissue could be a limiting factor. The homogenized sample had the highest yield (9.35ug), suggesting this may be a more efficient approach to obtaining gDNA (faster procedure and higher yields).
These results also demonstrate that gDNA can be successfully isolated from samples stored in RNA Later (gel pending).
Due to low yields of the R51 11 and R51 01 samples, these will not be run on a gel until the rest of the DNA is isolated.
Isolated the remainder of the gDNA from R51 11 and R51 01. Added an additional 0.5mL of DNAzol to each tube and pipetted up and down to further dissolve the remaining tissue. Then, proceeded as described above. Samples were resuspended in 200uL of 8mM NaOH with 2.02uL of 1M HEPES added.
Results:
Both samples have great yields and excellent 260/280 ratios. Digestion, combined with gentle pipetting to disrupt undigested tissue, appears to lead to higher yields. However, the process is more time consuming than just basic, physical homogenization of tissue.
Gel Loading (0.5ug of each DNA was loaded in a volume of 25uL)
Lane 1. Hyperladder
Lane 2. R51 11
Lane 3. R51 01
Lane 4. R51 01 homogenized
The R51 11 sample looks perfect. The other two samples however, appear degraded, with the homogenized sample looking the worst. Interestingly, those are both from the same source tissue, possibly suggesting that the DNA in this particular gill sample is actually degraded and is independent of the preparation, since the R51 11 (Lane 2) and R51 01 (Lane 3) were prepared simultaneously and in the same way. The gel also seems to show that physically homogenizing the sample leads to greater degradation than performing the Proteinase K digestion.
Set up gDNA isolation from the following samples:
R51 01 (WB R051-0410-01)
R51 11 (WB R051-0410-11)
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 O/N @ RT with rotation.
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.
Isolated RNA from 7 sepia samples received 20091125. Samples were removed from RNA Later, blotted and homogenized in 500uL of TriReagent. 500uL of additional TriReagent was added to the tubes after homogenization. Procedure was followed normally. The sepia retina RNA was isolated separately from the other samples and was resuspended in 100uL of 0.1% DEPC-H2O. The remaining samples were isolated and resuspended in 20uL of 0.1% DEPC-H2O. Nearly all samples had some sort of purple tint to them, ranging from almost black to extremely faint purple hue. The samples were spec’d and then stored @ -80C in Sam’s RNA Box #1.
Results:
Nearly all of the samples exhibited very strange curves and mediocre 260/280 ratios (for RNA). Could be due to the “purple stuff” carryover or possibly an effect of the RNA Later from which the samples were stored.
Received samples from Scott Lindell today. Two Ziplock bags taped together labelled “11/16/09 Clams scudders.” The bags contain 2mL screw cap tubes with small tissue samples in RNA later. One group of tubes is labelled with FL-3 # and the other group with BX-4 #. Samples will be stored at 4C to be processed later this month.
