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)

Back to Calendar

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
4. Measure Concentration in Nanodrop (different from before)