Revision as of 00:38, 23 July 2013 by >Djacobse
S2 Probe Preparation
Back to Calendar
Despite mixed reviews from the S4 set, I'm going to go ahead and make the S2 set. Also, I talked with Alan and he and I are going to prep some cells tomorrow. I'll probably go ahead and attempt fish with the S3 set, since that one worked out great. If I have other probes available, I may use them as well, but Alan and I won't be preparing the cells until tomorrow afternoon, which gives me plenty of time to prep a new set.
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 x17
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:2013-7-22-S2.png
Nanodrop Results
Sample
|
V4S4A
|
V4S4B
|
V4S4C
|
V4S4D
|
V6S4A
|
V6S4B
|
V6S4C
|
V6S4D
|
ng/uL
|
52.6
|
44.5
|
52.1
|
47.4
|
105.7
|
111.3
|
98.1
|
103.3
|
ug in 40 uL
|
2.1
|
1.8
|
2.1
|
1.9
|
4.2
|
4.5
|
3.9
|
4.1
|
Total ug
|
|
|
|
5.8
|
|
|
|
12.5
|
DpnII Digestion
1. Prepare Samples
Sample
|
V4S4A
|
V4S4B
|
V4S4C
|
V4S4D
|
V6S4A
|
V6S4B
|
V6S4C
|
V6S4D
|
ng/uL
|
52.6
|
44.5
|
52.1
|
47.4
|
105.7
|
111.3
|
98.1
|
103.3
|
ug in 35 uL
|
2.1
|
1.8
|
2.1
|
1.9
|
4.2
|
4.5
|
3.9
|
4.1
|
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 7/23/2013