Tag Archives: 5-azacitidine

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.

Share

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

Share

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.

Share

gDNA Isolation – Mac gigas larvae samples: control larvae 6.7.10 and 5-aza tr larvae 6.7.10

Continued gDNA isolation of the above mentioned larvae samples that was started by Mac yesterday. Amount of larvae in tubes looked disproportionately large, relative to the amount of DNAzol used in the O/N Proteinase K digestion(~500uL) so I added and additional 500uL of DNAzol to each of the two samples and gently pipetted a few times to mix.

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 800uL 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:

Yields are very good and the 260/280 ratios are pretty good. The 260/230 ratios are very poor and is likely due to the large amount of larvae used in the procedure. Will run samples on a gel to evaluate DNA integrity. UDPATE: Mac ran these samples on 6/9/10 (see her notebook on that date) and they look perfect.

Share