Daniel:Notebook/HiResChrPaint/2013-5-6

From ZhangLabWiki
Jump to navigation Jump to search

Probe Prep Test (Started 4/22/2013)[edit]

Back to Calendar

Column Purification vs. Ethanol Precipitation[edit]

Noi gave me her results today.  She got a similar yield from the PCR and from the DpnII digestion and lost quite a bit of DNA
in the lambda exonuclease digestion.  Of course we expect to lose ~50% (due to exonuclease eating a whole strand), but our purifications
post-digestion also show a high loss.  I'm going to try, with a large enough single-stranded sample, to compare EtOH precipitation with
the Zymo ssDNA column I bought a while back.
Noi has given me a set of primers ~50 bp.  She says that it is ~1400 ng/uL (~30 uL).  I'm going to use this to test the sample.

Column Purification[edit]

1. Prepared two samples: one with 1 uL (~1.4ug) and one with 2 uL primer (~3 ug). 
2. I added nf H20 to make 50 uL, then proceeded with the Zymo prep.
3. Refrigerated the samples to use later for comparison.

Ethanol Precipitation[edit]

1. Prepared four samples
uL sample 1 2 1 2
incubation at -80C 24 hr 24 hr 48 hr 48 hr
2. Added 300 uL nf H20 to each.
3. Added 900 uL 100% EtOH, 30 uL 3M NaOAc, and 1 uL glycoblue to each
4. Incubated at -80C for appropriate times