Daniel:Notebook/HiResChrPaint/2013-6-21
S1 Probe Preparation (Started 6/20/2013)
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