Daniel:Notebook/HiResChrPaint/2013-6-21

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S1 Probe Preparation (Started 6/20/2013)[edit]

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DpnII Digestion (Continued from 6/20/2013)[edit]

3. Pooled all V4 samples (4->1) and 2 V6 samples (4->2)
4. Column purification, elute with 40 uL EB
5. Measure Concentration in Nanodrop
6. TBE gel to ensure proper digestion

Nanodrop Results[edit]

Sample V4S1 V6S1AB V6S1CD
ng/uL 66.7 118.3 120.6
ug in 40 uL 2.7 4.7 4.8

TBE gel[edit]

1. Prepared samples 5.5 uL TBE, 0.5 uL sample, 2 uL 6x dye
2. Sample matrix
Lane 4 5 6 7 8 9
Sample 25 bp ladder V4S1B Undigested V6S1B Undigested V4S1 V6S1AB V6S1CD
3. Ran gel for 20 minutes at 220 V, stained for 2 minutes with 3 uL SYBR gold

Gel Image[edit]

File:DpnIIDigest 062113.jpg

Lambda Exonuclease digestion[edit]

1. Reaction table
Sample V4S1 V6S1AB V6S1CD
ng/uL 66.7 118.3 120.6
ug in 40 uL 2.7 4.7 4.8
uL 10x buffer 5 5 5
uL Lambda Exonuclease 3 5 5
uL nfH2O 7 5 5
total 50 50 50
3. Incubate at 37C for 1 hour followed by 10 minute heat shock at 75C
4. ssDNA column purification, elute with 10 uL

TBU gel[edit]

1. Sample Matrix; followed Protocol 2
Lane 3 4 5 6 7 8
Sample 0.5 uL LM ladder 1 uL LM ladder V4 (1:10) V6 (1:10) V4 (1:20) V6 (1:20)
uL sample 0.5 1 0.5 0.5 2* 2**
uL TBE 1.5 1 9.5 9.5 1 1
uL 2X Urea Buffer 2 2 10 10 1 1
Loaded 4 4 4 4 4 4

Note: *denotes sample taken from lane 5 sample; **denotes sample taken from lane 6 sample

2. Ran gel for 15 minutes at 250V
3. Stained with 3 uL SYBR gold for 10 minutes

TBU gel Results[edit]

Volume Report: File:GelQuant 062113.xls

File:GelQuant062113.jpg

File:GelQuant062113 Volume.jpg

Sample V4S1 V6S1
~ng/uL 60 140
ug in 10 uL 0.6 1.4