Revision as of 00:21, 21 June 2013 by >Djacobse
S1 Probe Preparation[edit]
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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
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 2 hours; resuspend in 50 uL nfH20
7. Column Purification, elute with 40 uL EB
8. Measure concentrations in nanodrop
qPCR Results[edit]
File:V4S1-062013.png
Nanodrop Results[edit]
Sample
|
V4S1A
|
V4S1B
|
V4S1C
|
V4S1D
|
V6S1A
|
V6S1B
|
V6S1C
|
V6S1D
|
ng/uL
|
30.7
|
31.1
|
31.5
|
29.1
|
90
|
87.7
|
91.9
|
89.9
|
ug in 40 uL
|
1.228
|
1.244
|
1.26
|
1.164
|
3.6
|
3.508
|
3.676
|
3.596
|
Total ug
|
|
|
|
4.896*
|
|
|
|
14.38**
|
Notes: *1.7x yield (in ug) of qPCR of V4S1 using 800 uL starting material; ** 2.1x yield (in ug) of qPCR of V6S1 using 800 uL starting material
Discussion[edit]
Results were not so good, although they should have been expected (see the notes). From the results I am skeptical of my final yields once all is
said and done. Therefore, I will proceed to DpnII digestion with caution.
DpnII Digestion[edit]
1. Prepare Samples
Sample
|
V4S1A
|
V4S1B
|
V4S1C
|
V4S1D
|
V6S1A
|
V6S1B
|
V6S1C
|
V6S1D
|
ng/uL
|
30.7
|
31.1
|
31.5
|
29.1
|
90
|
87.7
|
91.9
|
89.9
|
ug in 35 uL
|
1.1
|
1.1
|
1.1
|
1.0
|
3.2
|
3.1
|
3.2
|
3.1
|
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]]