Daniel:Notebook/HiResChrPaint/2013-5-6
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Probe Prep Test (Started 4/22/2013)[edit]
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