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
|