Daniel:Notebook/HiResChrPaint/2013-6-21

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Revision as of 20:38, 21 June 2013 by >Djacobse (Created page with "=S1 Probe Preparation (Started 6/20/2013)= Back to Calendar ==DpnII Digestion (Continued from [...")
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S1 Probe Preparation (Started 6/20/2013)

Back to Calendar

DpnII Digestion (Continued from 6/20/2013)

3. Pooled all V4 samples (4->1) and 2 V6 samples (4->2)
4. Column purification, elute with 40 uL EB
5. Measure Concentration in Nanodrop
6. TBE gel to ensure proper digestion

Nanodrop Results

Sample V4S1 V6S1AB V6S1CD
ng/uL 66.7 118.3 120.6
ug in 40 uL 2.7 4.7 4.8

TBE gel

1. Prepared samples 5.5 uL TBE, 0.5 uL sample, 2 uL 6x dye
2. Sample matrix
Lane 4 5 6 7 8 9
Sample 25 bp ladder V4S1B Undigested V6S1B Undigested V4S1 V6S1AB V6S1CD
3. Ran gel for 20 minutes at 220 V, stained for 2 minutes with 3 uL SYBR gold

Gel Image

[[Image:]]

Lambda Exonuclease digestion

1. Reaction table
2. {| class="wikitable" <hiddentext>generated with de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion V1.8</hiddentext>

|- style="font-size:12pt;font-weight:bold" align="center" | width="96" height="15" | Sample | width="65" | V4S1 | width="65" | V6S1AB | width="65" | V6S1CD

|- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 66.7 | align="center" | 118.3 | align="center" | 120.6

|- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 40 uL | align="center" | 2.7 | align="center" | 4.7 | align="center" | 4.8

|- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL 10x buffer | align="center" | 5 | align="center" | 5 | align="center" | 5

|- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | uL Lambda Exonuclease | align="center" | 3 | align="center" | 5 | align="center" | 5

|- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL nfH2O | align="center" | 7 | align="center" | 5 | align="center" | 5

|- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | total | align="center" | 50 | align="center" | 50 | align="center" | 50

|}

3. Incubate at 37C for 1 hour followed by 10 minute heat shock at 75C
4. ssDNA column purification, elute with 10 uL