Daniel:Notebook/HiResChrPaint/2013-7-17: Difference between revisions
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==Lambda Exonuclease Digestion== | ==Lambda Exonuclease Digestion== | ||
1. Reaction table | |||
{| 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="101" height="15" | Sample | |||
| width="65" | V4S3A | |||
| width="65" | V4S3B | |||
| width="65" | V4S3C | |||
| width="65" | V4S3D | |||
| width="65" | V6S3A | |||
| width="65" | V6S3B | |||
| width="65" | V6S3C | |||
| width="65" | V6S3D | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ng/uL | |||
| align="center" | 84.9 | |||
| align="center" | 82.3 | |||
| align="center" | 85.4 | |||
| align="center" | 86.4 | |||
| align="center" | 107.6 | |||
| align="center" | 98.2 | |||
| align="center" | 107.4 | |||
| align="center" | 109.2 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ug in 25 uL | |||
| align="center" | 2.1 | |||
| align="center" | 2.1 | |||
| align="center" | 2.1 | |||
| align="center" | 2.2 | |||
| align="center" | 2.7 | |||
| align="center" | 2.5 | |||
| align="center" | 2.7 | |||
| align="center" | 2.7 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | uL added | |||
| align="center" | 25 | |||
| align="center" | 25 | |||
| align="center" | 25 | |||
| align="center" | 25 | |||
| align="center" | 25 | |||
| align="center" | 25 | |||
| align="center" | 25 | |||
| align="center" | 25 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | uL 10x buffer | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="45" | uL Lambda Exonuclease (5U/uL) | |||
| align="center" | 3 | |||
| align="center" | 3 | |||
| align="center" | 3 | |||
| align="center" | 3 | |||
| align="center" | 3 | |||
| align="center" | 3 | |||
| align="center" | 3 | |||
| align="center" | 3 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | uL nfH2O | |||
| align="center" | 17 | |||
| align="center" | 17 | |||
| align="center" | 17 | |||
| align="center" | 17 | |||
| align="center" | 17 | |||
| align="center" | 17 | |||
| align="center" | 17 | |||
| align="center" | 17 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | total | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
|} | |||
2. Incubate for 1 hour at 37C followed by 10 minute heat inactivation at 75C | |||
3. ssDNA column purification | |||
4. '''Measure Concentration in Nanodrop''' (different from before) |
Revision as of 19:18, 17 July 2013
S3 Probe Preparation (Started 7/16/2013)
DpnII Digestion
3. Column Purification, elute with 30 uL EB 4. Measure concentrations in nanodrop
Nanodrop Results
Sample | V4S3A | V4S3B | V4S3C | V4S3D | V6S3A | V6S3B | V6S3C | V6S3D |
ng/uL | 84.9 | 82.3 | 85.4 | 86.4 | 107.6 | 98.2 | 107.4 | 109.2 |
ug in 30 uL | 2.5 | 2.5 | 2.6 | 2.6 | 3.2 | 2.9 | 3.2 | 3.3 |
total ug | 10.2 | 12.7 |
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 | V4S3A | V4S3B | V4S3C | V4S3D | V6S3A | V6S3B | V6S3C | V6S3D |
2. Run for 25 minutes at 250V
Gel Image
Lambda Exonuclease Digestion
1. Reaction table
Sample | V4S3A | V4S3B | V4S3C | V4S3D | V6S3A | V6S3B | V6S3C | V6S3D |
ng/uL | 84.9 | 82.3 | 85.4 | 86.4 | 107.6 | 98.2 | 107.4 | 109.2 |
ug in 25 uL | 2.1 | 2.1 | 2.1 | 2.2 | 2.7 | 2.5 | 2.7 | 2.7 |
uL added | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
uL 10x buffer | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
uL Lambda Exonuclease (5U/uL) | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
uL nfH2O | 17 | 17 | 17 | 17 | 17 | 17 | 17 | 17 |
total | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 |
2. Incubate for 1 hour at 37C followed by 10 minute heat inactivation at 75C 3. ssDNA column purification 4. Measure Concentration in Nanodrop (different from before)