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

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.

MeDIP – SB/WB Fragmented gDNA (continued from yesterday)

Continued MeDIP process from yesterday. Protein A/G beads were pelleted XXXXXXXXX, supe transferred to clean tube. Beads were washed 3x in the following fashion, each wash saved to retain unmethylated DNA:

1.

Samples were phenol:chloroform extracted and EtOH precipitated:

  1. Added equal volume of phenol:chloroform:IAA, vortexed, spun @ 12,500g, 5mins, 4C.

  2. Transferred aqueous phase to clean tube.

  3. Added equal volume of chloroform, vortexed, spun @ 12,500g, 5mins, 4C.

  4. Transferred aqueous phase to clean tube.

  5. Added 0.1 vols 3M NaOAc (pH=5.2), 2.5 vols of 100% EtOH, mixed and stored @ -20C over the weekend.

Will finish precipitation next week and quantify recovery.

Restriction Digests – Various gigas gDNAs of Mac’s

Performed restriction digests. Made dilutions of all DNAs involved of 25ng/uL. Made enough for a total of 9 digests could be performed on each DNA. This allowed using 10uL of each DNA for each rxn, more mileage out of the lowest concentration sample (R37-01), and allowed for the use of master mixes when preparing the digests. All calculations/dilutions/master mixes can be seen here. Each DNA was digested individually with HpaII, MspI and undigested. Incubated the digests 4hrs @ 37C. After digestion, performed an EtOH precipitation. Added 0.1 vols of 3M NaOAc (pH=5.2), then 2.5 vols of 100% EtOH. Mixed by inversion and incubated 30mins @ -20C. Pelleted DNA 16,000g, 30mins @ 4C. Discarded supe. Washed pellets with 1mL 70% EtOH. Pelleted DNA 16,000g, 15mins, 4C. Discarded supe. Resuspended DNA in 10uL PCR H2O and spec’d.

Results:

Well, the recovery of DNA is very low. The best recovery is ~50% while the worst is around ~1%.

I did not proceed with the intended qPCR due to the low yields and the fact that I don’t know if we’ve previously tested how sensitive our assay(s) our for our target genes. Will discuss with Steven/Mac next week.

MeDIP – SB/WB Fragmented gDNA (continued from yesterday)

Continued MeDIP process from yesterday. Added 20uL of Protein A/G Plus Agarose (Santa Cruz Biotech) beads to each sample and continued incubation with rotation @ 4C for 2hrs. Pelleted the Protein A/G beads 3300g, 2mins, 4C.

Removed and saved supe (to retain unmethylated DNA). Washed beads with 1mL 1x MeDIP Buffer. Repeated two more times. Saved supe after each wash.

Resuspended beads in 250uL MeDIP Digestion Buffer (50mM Tris-HCl, pH=8.0, 10mM EDTA, pH=8.0, 0.5% SDS). Added 75ug of Proteinase K. Incubated 20hrs @ RT with end-over-end rotation.

Note: The protocol we have says to incubate the Proteinase K digest @ 55C. However, we don’t have a means to do so, since we need a rocker/rotator to keep the agarose beads in suspension. According to various sources, Proteinase K retains >80% of it’s enzymatic activity between 20C-50C. So, I’ve allowed the digest to run longer (24hrs) than recommended (O/N).

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.

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).

gDNA Sonication – SB/WB gDNA pools (prep for MeDIP) from 20100618

The previous attempt at sonication (see 20100618) failed, likely due to no using the correct equipment (tubes and Covaris adapter). The two gDNA pools, which had previously been unsuccessfully fragmented on 20100618 (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

AFA Intensifier tubes and associated Covaris adapter.

After shearing, ran 250ng of each pool on a 2% TAE agarose gel for fragmentation verification.

Results:

Lane 1 – Hyperladder I

Lane 2 – R37

Lane 3 – R51

Looking at this gel, the samples have been successfully fragmented and I would estimate have generated and average fragment size of ~400bp (going from bottom to top of the Hyperladder: 200bp, 400bp, 600bp, 800bp, 1000bp). So, this looks great! Can proceed with remainder of MeDIP procedure at any time.

Additionally, I will confirm a more accurate assessment of average fragment size by running these two samples on the Agilent Bioanalyzer.

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.