Revision as of 00:20, 17 July 2013 by >Djacobse
S3 Probe Preparation
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Since I don't have any S3 set left, I'm going to make a new run using the various new techniques, specifically I am more confident in the dye coupling stage now. I'm going to make just the aa-dUTP (ARES labeling) style for now, since it is difficult to balance the reactions when I have a lot of samples.
PCR Amplification
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
|
V4S3 (12.5x)
|
2.5
|
625
|
5
|
5
|
0
|
0
|
31
|
581.5
|
1250
|
V6S3 (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
vii. 72C 2 min
viii. 16C hold
4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total)
5. Ethanol Precipitation, added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue
6. Incubated 4 hours; resuspend in 50 uL nfH20
7. Column Purification, elute with 40 uL EB
8. Measure concentrations in nanodrop
qPCR Results
File:V4S3-071613.png
Nanodrop Results
Sample
|
V4S1A
|
V4S1B
|
V4S1C
|
V4S1D
|
V6S1A
|
V6S1B
|
V6S1C
|
V6S1D
|
ng/uL
|
97.6
|
91.6
|
99
|
99.8
|
118.6
|
113.8
|
117.7
|
118.8
|
ug in 40 uL
|
3.9
|
3.7
|
4.0
|
4.0
|
4.7
|
4.6
|
4.7
|
4.8
|
Total ug
|
|
|
|
15.5
|
|
|
|
18.8
|
DpnII Digestion
1. Prepare Samples
Sample
|
V4S3A
|
V4S3B
|
V4S3C
|
V4S3D
|
V6S3A
|
V6S3B
|
V6S3C
|
V6S3D
|
ng/uL
|
97.6
|
91.6
|
99
|
99.8
|
118.6
|
113.8
|
117.7
|
118.8
|
ug in 35 uL
|
3.4
|
3.2
|
3.5
|
3.5
|
4.2
|
4.0
|
4.1
|
4.2
|
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 16 hours; 20 minute heat shock at 65C; Continued 6/21/2013]]