Daniel:Notebook/HiResChrPaint/2013-7-23

From ZhangLabWiki
Revision as of 22:00, 23 July 2013 by >Djacobse
Jump to navigation Jump to search

S2 Probe Preparation (Started 7/22/2013)

Back to Calendar

DpnII Digestion (from 7/22/2013)

3. Pool samples for V4 set into 4AB and 4CD
4. Column Purification, elute with 30 uL EB**
5. Pooled 4AB and 4CD into V4S2 and pooled V6 sets into V6AB and V6CD
6. Measure concentrations in nanodrop

Note (**): Forgot to move the columns to fresh tubes before elution. Caught myself just as the centrifuge started, so I stopped it immediately, before it even got to full speed. Still, this resulted in me losing most of the samples (I also threw away the collection tubes before proceeding to the fresh collection tube).

Nanodrop Results

Sample V4S2 V6S2AB V6S2CD
ng/uL 142.9 32.8 34.9
ug in 20 uL 2.9 0.7 0.7
total ug     1.4

DpnII Digest gel

1. Lanes; (5.5 uL TBE; 2 uL 6x buffer, 0.5 uL sample; load 7 uL for all)


Lane 1 2 3 4 5 6 7 8 9 10 11 12
Sample 25bp ladder V4S2 V6S2AB V4S2CD

Gel Image

File:DpnIIDigest 72313.jpg

Discussion

Since I botched this probe prep, I may have to start over. I still have to digest with lambda exonuclease, which is going to drop my concentrations by 50% regardless. This would mean I'd likely have <1 ug for both V4S2 and V6S2 prior to dye coupling. It's just not worth it. I'm going to store these probes, and the next time I make a S2 set I'll add the probes in post DpnII digestion.

S2 Probe Prep