Daniel:Notebook/HiResChrPaint/2013-7-17: Difference between revisions
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==Dye Coupling== |
Revision as of 22:14, 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 | 10bp 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. Pool samples 4. ssDNA column purification, elute with 20 uL 5. Measure Concentration in Nanodrop (different from before) 6. Dry samples in vaccuum centrifuge
Nanodrop Results
Sample | V4S3 | V6S3 |
ng/uL | 166 | 228.1 |
ug in 20 uL | 3.3 | 4.6 |