Tag Archives: EtOH precipitation

EtOH Precipitations – HpaII and MspI 2nd Round Digests from 20101124

Samples were EtOH precipitated, according to protocol. Samples were resuspended in 20uL of Qiagen’s EB and spec’d.

Samples are labeled as Parent (P), #, tissue, enzyme (MspI = M, HpaII = H, Undigested = U)

Results:

Spreadsheet of spec values is here. Overall, poor recoveries from all of the digested samples, but decent recoveries from the undigested samples. The samples were passed to Mac who performed qPCR using two different primer sets. Please see her notebook for the results of the qPCR.

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Phenol:Chloroform Extractions and EtOH Precipitations – HapII and MspI digests from yesterday

Restriction digests from yesterday were mixed with equal volume (50uL) of phenol:chloroform:IAA (25:24:1) and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to a clean tube and an equal volume (50uL) of chloroform was added. Samples were mixed and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to clean tubes and EtOH precipitated, according to protocol. Samples were resuspended in 25uL of H2O and spec’d.

Samples are labeled as Parent (P), #, tissue, enzyme (MspI = M, HpaII = H, Undigested = U)

Results:

Here is a link to a spreadsheet with ODs. Average recovery was ~734ng, which is only a 36% recovery (started with 2000ng). Will need to discuss with Mac and Steven to see if it’s worth continuing with these sample through a second round of digests/phenol:chloroform extraction/EtOH precipitation, as I don’t know what quantity of DNA is required/desired for the subsequent methylation specific PCR (MSP), OR if I should/need to perform a repeat of these 1st-round digestions in order to end up with sufficient DNA for MSP.

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EtOH Precipitation – Whale gDNA from 20101022

Precipitated whale gDNA in hopes of producing a sample with a higher concentration. Added 0.1 vols of 3M NaOAc (10uL), 2.5 vols of 100% EtOH (275uL), mixed thoroughly and incubated @ -20C for 1hr. DNA was pelleted by spinning sample @ 16,000g, 30mins, 4C. No visible pellet. Supe was removed, sample was washed with 1mL 75% EtOH, and pelleted by spinning @ 16,000g, 15mins, 4C. Supe was removed, sample resuspended in 10uL nuclease-free H2O and spec’d.

Results:

No DNA to speak of (spec data not shown).

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EtOH Precipitation – Hard Clam RNA from earlier today

RNA was mixed with 0.1 vols of 3M NAOAc (pH = 5.2) and 2.5 vols of 100% EtOH, vortexed and incubated @ -20C for 30mins. RNA was pelleted @ 16,000g, 30mins, 4C. Supe was discarded and pellet was washed with 1mL 70% EtOH. RNA was pelleted @ 16,000g, 15mins, 4C. This was step was repeated a second time. Supe was discarded, the RNA was resuspended in 50uL of 0.1% DEPC-H2O, spec’d and stored @ -80C:

Results:

Interestingly, precipitating the samples vastly improved the 260/230 ratios. However, the 260/280 ratios DECREASED for all samples except one (CA 1). Not really thrilled about this fact, nor am I sure why this would happen.

RNA was stored @ -80C in “Sam’s RNA Box #1.”

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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 (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.

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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.

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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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RNA Precipitation and Fragmentation for SOLiD Libraries – Pooled abalone mRNA (from yesterday)

mRNA was precipitated according to Ambion’s MicroPolyA Purist Kit protocol. Added 0.1vols of ammonium acetate, 2.5vols of 100% EtOH and incubated 30mins @ -80C. Samples were pelleted, washed with 1mL 70% EtOH, pelleted, resuspended in 8uL of nuclease-free H2O and spec’d:

After precipitation, samples were fragmented with RNase III according to the Ambion Whole Transcriptome Analysis Kit protocol and then cleaned up using the Invitrogen Ribominus Concentration Module, according to the Ambion Whole Transcriptome Analysis Kit protocol. 0.5uL of each sample was removed for analysis on the Bioanalyzer.

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mRNA Precipitation – Herring Liver mRNA for SOLiD Libraries (continued from yesterday)

Spun samples 16,000g, 30mins, 4C. Discarded supe, quick spun tubes, removed residual supe, washed with 1mL 70% EtOH. Spun samples 16,000g, 15mins, 4C. Discarded supe, quick spun tubes, removed residual supe, resuspended in 8.5uL of nuclease-free H2O. Stored @ -80C until ready to proceed with fragmentation for SOLiD libraries.

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