Daniel:Notebook/HiResChrPaint/2013-6-21: Difference between revisions
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[[Image:GelQuant062113_Volume.jpg|600px]] | [[Image:GelQuant062113_Volume.jpg|600px]] | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-weight:bold" align="center" | |||
| width="69" height="12" | Sample | |||
| width="69" | V4S1 | |||
| width="69" | V6S1 | |||
|- align="center" | |||
|style="font-weight:bold" height="12" | ~ng/uL | |||
| align="center" | 60 | |||
| align="center" | 140 | |||
|- align="center" | |||
|style="font-weight:bold" height="12" | ug in 10 uL | |||
| align="center" | 0.6 | |||
| align="center" | 1.4 | |||
|} |
Latest revision as of 23:09, 21 June 2013
S1 Probe Preparation (Started 6/20/2013)[edit]
DpnII Digestion (Continued from 6/20/2013)[edit]
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[edit]
Sample | V4S1 | V6S1AB | V6S1CD |
ng/uL | 66.7 | 118.3 | 120.6 |
ug in 40 uL | 2.7 | 4.7 | 4.8 |
TBE gel[edit]
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[edit]
File:DpnIIDigest 062113.jpg
Lambda Exonuclease digestion[edit]
1. Reaction table
Sample | V4S1 | V6S1AB | V6S1CD |
ng/uL | 66.7 | 118.3 | 120.6 |
ug in 40 uL | 2.7 | 4.7 | 4.8 |
uL 10x buffer | 5 | 5 | 5 |
uL Lambda Exonuclease | 3 | 5 | 5 |
uL nfH2O | 7 | 5 | 5 |
total | 50 | 50 | 50 |
3. Incubate at 37C for 1 hour followed by 10 minute heat shock at 75C 4. ssDNA column purification, elute with 10 uL
TBU gel[edit]
1. Sample Matrix; followed Protocol 2
Lane | 3 | 4 | 5 | 6 | 7 | 8 |
Sample | 0.5 uL LM ladder | 1 uL LM ladder | V4 (1:10) | V6 (1:10) | V4 (1:20) | V6 (1:20) |
uL sample | 0.5 | 1 | 0.5 | 0.5 | 2* | 2** |
uL TBE | 1.5 | 1 | 9.5 | 9.5 | 1 | 1 |
uL 2X Urea Buffer | 2 | 2 | 10 | 10 | 1 | 1 |
Loaded | 4 | 4 | 4 | 4 | 4 | 4 |
Note: *denotes sample taken from lane 5 sample; **denotes sample taken from lane 6 sample
2. Ran gel for 15 minutes at 250V 3. Stained with 3 uL SYBR gold for 10 minutes
TBU gel Results[edit]
Volume Report: File:GelQuant 062113.xls
File:GelQuant062113 Volume.jpg
Sample | V4S1 | V6S1 |
~ng/uL | 60 | 140 |
ug in 10 uL | 0.6 | 1.4 |