Tag Archives: R51

MeDIP – SB/WB Fragmented gDNA EtOH precipitation (continued from 20100702)

Finished EtOH precipitation of MeDIP gDNA. Samples were pelleted 16,000g, 4C, 30mins. Supe was discarded. Washed with 1mL 70% EtOH, pelleted 16,000g, 4C, 15mins. Supe discarded. MeDIP DNA was resuspended in 100uL of TE (pH = 8.5). Wash samples, containing unmethylated DNA, were resuspended/combined in a total of 100uL TE (pH = 8.5). Samples were spec’d:

Results:

R37: MeDIP DNA = 1.393ug recovery. This is ~13% of the input total gDNA (11.25ug) and is ~28% of the total DNA recovered in the procedure (4.935ug). Unmethylated DNA = 3.542ug total recovery. This is ~31% of the input total gDNA (11.25ug) and is ~72% of the total DNA recovered in the procedure (4.935ug). Total DNA recovery = ~44%.

R51: MeDIP DNA = 1.256ug recovery. This is ~14% of the input total gDNA (8.75ug) and is ~23% of the total DNA recovered in the procedure (5.462ug). Unmethylated DNA = 4.206ug total recovery. This is ~48% of the input total gDNA (8.75ug) and is ~77% of the total DNA recovered in the procedure (5.462ug). Total DNA recovery = ~62%.

There definitely seemed to be a high degree of salt carryover from the procedure, despite the phenol:chloroform treatment and EtOH precipitation. As such, I believe this is the reason that the 260/230 ratios are so out of whack. Possibly explains why the 260/280 ratios for the MeDIP DNA are so high, too?

These results demonstrate what we can expect to recover from this procedure, as well as how much DNA gets lost during processing. MeDIP DNA and unmethylated DNA were stored @ -20C.

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MeDIP – SB/WB Fragmented gDNA (from 20100625)

After confirming proper fragmentation (~460bp average fragment size) via Bioanalyzer earlier today, began the MeDIP process. Brought fragmented DNA samples up to 350uL with TE. Heated samples @ 95C for 10mins, then incubated on ice 5mins. Added 100uL of 5x MeDIP Buffer (50mM Na2HPO4, 700mM NaCl, 0.25% Triton-X 100), 45uL of TE and 5uL (5ug) of anti-methyl cytidine antibody (Diagenode; 5-mC monoclonal antibody cl. b). Incubated O/N, 4C rotating.

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Bioanalyzer – Fragmented SB/WB gDNA (from 20100625)

To gain a more quantitative assessment of the fragmentation from 20100625, I ran 1uL of each sample (~55ng, according to pre-fragmentation spec values) on the Agilent Bioanalyzer 2100, using the DNA 1000 kit, according to manufacturer’s protocol.

Results:

Avg. size of fragmentation is ~460bp for the two samples. Fragmentation size was determined by marking the same region on both sample’s electropherograms (see below).

R37: Avg. size = 450bp (in Region 1, marked with blue lines in image below)

 

 

R51: Avg. size = 468bp (in Region 1, marked with blue lines in image below)

 

 

 

Overlay of R37 and R51 fragmentation. Note that both electropherograms are nearly identical (this is good).

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gDNA Sonication – SB/WB gDNA pools (prep for MeDIP) from earlier today

The two gDNA pools (SB and WB) were sonicated using a Covaris S2. Used the guidelines of the manufacturer (listed below) for shearing gDNA to a desired target size (500bp):

Duty Cycle: 5%

Intensity: 3

Cycels per Burst: 200

Time (seconds): 90

Temp (water bath): 4C

Power Mode: Frequency Sweeping

Sample Volume: 120uL

Buffer: TE

DNA Mass: ~8ug

Starting Material: >50kb

To be noted, the Covaris guidelines list the use of an “AFA Intensifier” tube, which I did not use (because we don’t have them).

After shearing, ran 250ng of each pool on a 2% TAE agarose gel for fragmentation verification. Also ran 250ng of pre-sonication DNA from each pool as controls.

Results:

Lane 1 – Hyperladder I

Lane 2 – R37, Un-sonicated

Lane 3 – R37, sonicated

Lane 4 – R51, Un-sonicated

Lane 5 – R51, sonicated

Sonication with the Covaris did NOT produce the desired fragmentation (500bp smear) in either sample, although the R37 sonicated samples shows a significantly greater degree of fragmentation than the R51 sonicated sample. Not sure how to explain this difference, other than the R51 sample has a greater amount of DNA. Additionally, the results could be explained by the fact that we did not use the AFA Intensifier listed in the Covaris guidelines…

Am consulting with a person in Genome Sciences who has used a Covaris for DNA fragmentation in the past to see if the AFA Intensifiers are indeed necessary and, if so, we can use two of them. Hopefully have an answer soon and be able to proceed with additional fragmentation next week.

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gDNA Precipitation – SB/WB gDNA pools (prep for MeDIP)

8 gDNA samples from SB were pooled and 8 gDNA samples from WB were pooled, using equal amounts of gDNA from each sample (1250ng) for a total of 10ug (see SB/WB Mac’s MeDIP spreadsheet for specific samples/volumes used in pooling). Since samples were stored in pH-adjusted NaOH (see 20100605), they needed to be precipitated in order to have the gDNA suspended in TE for the downstream steps of methylated DNA immunoprecipitation (MeDIP). 10% 3M sodium acetate (pH = 5.2) was added to each tube, then 2.5 vols of 100% EtOH and mixed. Samples were incubated @ -20C for 30mins. DNA was pelleted by spinning 16,000g for 30mins @ 4C. Supe was discarded. Pellets were washed with 1mL 70% EtOH and then pelleted @ 16,000g for 10mins @ 4C. Supe was discarded and gDNA was resuspended in 120uL of TE (pH = 8.0) and spec’d.

Results:

The R37 (SB) sample pool yielded 7.056ug after precipitation and the R51 (WB) sample pool yielded 8.834ug after precipitation (started with 10ug). This is good, as 6ug is needed for MeDIP and I wanted to have some (~250ng) available for running as an un-sonicated control on the post-sonication gel. Will transfer 250ng from each pool to separate tubes and then proceed with sonication.

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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.

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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.

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gDNA Isolation – Mac gigas gill samples (continued from yesterday)

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.

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gDNA Isolation – Mac gigas gill samples

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.

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