Revision as of 00:37, 3 August 2013 by >Djacobse
S1 Probe Preparation[edit]
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Armed with a new bottle of water (seriously...), I'm now ready to amplify the S1 set.
PCR Amplification[edit]
1. Reaction Table
|
20 nM OligoPool
|
2X Kapa SYBR qPCR MM
|
100 uM AP1V41U
|
100 uM AP2V4
|
100 uM AP1V61U
|
100 uM AP2V6
|
2 mM aa-dUTP
|
H20
|
Total Volume
|
per rxn
|
0.2
|
50
|
0.4
|
0.4
|
0.4
|
0.4
|
2.5
|
45.7
|
100
|
V4S1 (12.5x)
|
2.5
|
625
|
5
|
5
|
0
|
0
|
31
|
581.5
|
1250
|
V6S1 (12.5x)
|
2.5
|
625
|
0
|
0
|
5
|
5
|
31
|
581.5
|
1250
|
2. Aliquot 100 uL into each of 12 qPCR tubes
3. PCR protocol:
i. PCR 3 min 95C
ii. 95C 3 sec
iii. 55C 30 sec
iv. 72C 20 sec
v. plate read
vi. goto ii x17
vii. 72C 2 min
viii. 16C hold
4. Pool 3 samples together each (300 uL each, 4 pools per set, 8 total)
5. Column Purification, elute with 40 uL EB
6. Measure concentrations in nanodrop
qPCR Results[edit]
Nanodrop Results[edit]
Sample
|
V4S1A
|
V4S1B
|
V4S1C
|
V4S1D
|
V6S1A
|
V6S1B
|
V6S1C
|
V6S1D
|
ng/uL
|
96.8
|
92.3
|
90.1
|
97.2
|
141.6
|
148.1
|
142
|
136.3
|
ug in 40 uL
|
3.9
|
3.7
|
3.6
|
3.9
|
5.7
|
5.9
|
5.7
|
5.5
|
Total ug
|
|
|
|
15.1
|
|
|
|
22.7
|
DpnII Digestion[edit]
1. Prepare Samples
Sample
|
V4S1A
|
V4S1B
|
V4S1C
|
V4S1D
|
V6S1A
|
V6S1B
|
V6S1C
|
V6S1D
|
ng/uL
|
96.8
|
92.3
|
90.1
|
97.2
|
141.6
|
148.1
|
142
|
136.3
|
ug in 35 uL
|
3.4
|
3.2
|
3.2
|
3.4
|
5.0
|
5.2
|
5.0
|
4.8
|
uL 10X buffer
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
uL DpnII (50U/uL)
|
2
|
2
|
2
|
2
|
2
|
2
|
2
|
2
|
uL nfH2O
|
8
|
8
|
8
|
8
|
8
|
8
|
8
|
8
|
Total
|
50
|
50
|
50
|
50
|
50
|
50
|
50
|
50
|
2. Incubate samples at 37C for 2 hours; 20 minute heat shock at 65C
3. Column purification, elute with 30 uL EB
4. Measure in Nanodrop
Nanodrop Results[edit]
Sample
|
V4S1A
|
V4S1B
|
V4S1C
|
V4S1D
|
V6S1A
|
V6S1B
|
V6S1C
|
V6S1D
|
ng/uL
|
88.2
|
87.1
|
77.4
|
94.7
|
129.9
|
142.6
|
139.6
|
132.3
|
ug in 30 uL
|
2.6
|
2.6
|
2.3
|
2.8
|
3.9
|
4.3
|
4.2
|
4.0
|
Total ug
|
|
|
|
10.4
|
|
|
|
16.3
|
TBE Gel[edit]
Lane
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
10
|
11
|
12
|
Sample
|
|
|
10 bp ladder
|
V4S1A
|
V4S1B
|
V4S1C
|
V4S1D
|
V6S1A
|
V6S1B
|
V6S1C
|
V6S1D
|
|
Gel Image