Daniel:Notebook/HiResChrPaint/2013-7-17

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Revision as of 19:19, 17 July 2013 by >Djacobse
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S3 Probe Preparation (Started 7/16/2013)

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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

File:DpnIIDigest 071713.jpg

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, elute with 10 uL
4. Measure Concentration in Nanodrop (different from before)