Daniel:Notebook/HiResChrPaint/2013-9-4: 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="65" height="15" | Sample
| width="65" | BSPP 1AB
| width="65" | BSPP 1CD
| width="65" | BSPP 2AB
| width="65" | BSPP 2CD
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 127.7
| align="center" | 130.6
| align="center" | 110.1
| align="center" | 109.3
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 25 uL
| align="center" | 3.2
| align="center" | 3.3
| align="center" | 2.8
| 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
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="30" | uL 10x buffer
| 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
|- 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
|- 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
|}
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

Revision as of 21:35, 4 September 2013

BSPP Control (Started 09/03/2013)

Back to Calendar

DpnII Digestion (Started 09/03/2013)

3. Column Purification, elute with 30 uL EB
4. Measure concentrations in nanodrop

Nanodrop Results

Sample BSPP 1AB BSPP 1CD BSPP 2AB BSPP 2CD
ng/uL 127.7 130.6 110.1 109.3
ug in 30 uL 3.8 3.9 3.3 3.3
total   7.7   6.6

TBE 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 BSPP 1AB BSPP 1CD BSPP 2AB BSPP 2CD
2. Run for 30 minutes at 200V
3. Stain with 3 uL SYBR gold for 2 minutes

Gel Image

File:2013-09-04-DpnIIDigest-BSPP.jpg


The gel looks fine, so I'm going to proceed as usual.

Lambda Exonuclease Digestion

1. Reaction table
Sample BSPP 1AB BSPP 1CD BSPP 2AB BSPP 2CD
ng/uL 127.7 130.6 110.1 109.3
ug in 25 uL 3.2 3.3 2.8 2.7
uL added 25 25 25 25
uL 10x buffer 5 5 5 5
uL Lambda Exonuclease (5U/uL) 3 3 3 3
uL nfH2O 17 17 17 17
total 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