Strand Control[edit]
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Andrew has pointed out to me that I should always do the processing on the probes, just to keep results consistent. I am fairly confident this is not the cause of my difficulties, but it is still wise to keep that particular aspect consistent. As such, I will be running through the more rigorous method of probe prep.
I'll be running through V6S2 and V6S3, my favorites.
- Reaction Table
|
10 nM OligoPool
|
2X Kapa SYBR qPCR MM
|
100 uM AP1V61U
|
100 uM AP2V6
|
H20
|
Total Volume
|
per rxn
|
0.4
|
50
|
0.4
|
0.4
|
48.8
|
100
|
V6S2 (12.2x)
|
4.88
|
610
|
4.88
|
4.88
|
595.36
|
1220
|
V6S3 (12.2x)
|
4.88
|
610
|
4.88
|
4.88
|
595.36
|
1220
|
- Aliquot 100 uL into each of 12 qPCR tubes
- PCR protocol:
- PCR 3 min 95C
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto ii x17
- 72C 2 min
- 16C hold
- Pool 3 samples together
- Column Purification, elute with 40 uL EB
- Measure concentrations in nanodrop
qPCR Results[edit]
This is a caption.
Nanodrop Results
|
V6S2A
|
V6S2B
|
V6S2C
|
V6S2D
|
V6S3A
|
V6S3B
|
V6S3C
|
V6S3D
|
ng/uL
|
99.9
|
95.4
|
94.6
|
84.5
|
110
|
112.2
|
111.6
|
99.8
|
ug in 40 uL
|
4.0
|
3.8
|
3.8
|
3.4
|
4.4
|
4.5
|
4.5
|
4.0
|
ug total
|
|
|
|
15.0
|
|
|
|
17.3
|
|
|
|
|
|
|
|
|
|
DpnII Digestion[edit]
- Reaction Table
|
V6S2A
|
V6S2B
|
V6S2C
|
V6S2D
|
V6S3A
|
V6S3B
|
V6S3C
|
V6S3D
|
ng/uL
|
99.9
|
95.4
|
94.6
|
84.5
|
110
|
112.2
|
111.6
|
99.8
|
ug in 35 uL
|
3.5
|
3.3
|
3.3
|
3.0
|
3.9
|
3.9
|
3.9
|
3.5
|
uL sample
|
35
|
35
|
35
|
35
|
35
|
35
|
35
|
35
|
uL 10X buffer
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
uL nfH20
|
8
|
8
|
8
|
8
|
8
|
8
|
8
|
8
|
uL DpnII (10 U/uL)
|
2
|
2
|
2
|
2
|
2
|
2
|
2
|
2
|
Total
|
50
|
50
|
50
|
50
|
50
|
50
|
50
|
50
|
- Incubate 16 hours at 37C
- Heat inactivate 20 minutes at 65C
- Continued tomorrow