Positive Control 2[edit]
Back to Calendar
I'm going to start my work on positive control 2 today, amplifying all 4 probe sets and eventually coupling them to a single dye (Alexa 488). Since I've found I work best only amplifying 2 sets at a time, I'll amplify V6S1 and V6S2 first, and amplify S3 and S4 starting tomorrow or Saturday.
qPCR Amplification[edit]
1. Reaction table
|
10 nM OligoPool
|
2X Kapa SYBR qPCR MM
|
100 uM AP1V61U-Amino
|
100 uM AP2V6
|
H20
|
Total Volume
|
per rxn
|
0.4
|
50
|
0.4
|
0.4
|
48.8
|
100
|
V6S1 (12.5x)
|
5
|
625
|
5
|
5
|
610
|
1250
|
V6S2 (12.5x)
|
5
|
625
|
5
|
5
|
610
|
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. Column Purification, elute with 40 uL EB
6. Measure concentrations in nanodrop
qPCR Results[edit]
File:2013-11-14-V6S1-V6S2.png
Nanodrop Results[edit]
Probe
|
V6S1A
|
V6S1B
|
V6S1C
|
V6S1D
|
V6S2A
|
V6S2B
|
V6S2C
|
V6S2D
|
ng/uL
|
81.5
|
81.2
|
80.1
|
79.2
|
90.1
|
84.9
|
101.2
|
88
|
ug in 40 uL
|
3.3
|
3.2
|
3.2
|
3.2
|
3.6
|
3.4
|
4.0
|
3.5
|
Total
|
|
|
|
12.9
|
|
|
|
14.6
|
DpnII Digestion[edit]
1. Reaction Table
Sample
|
V6S1A
|
V6S1B
|
V6S1C
|
V6S1D
|
V6S2A
|
V6S2B
|
V6S2C
|
V6S2D
|
ng/uL
|
81.5
|
81.2
|
80.1
|
79.2
|
90.1
|
84.9
|
101.2
|
88
|
ug in 35 uL
|
2.9
|
2.8
|
2.8
|
2.8
|
3.2
|
3.0
|
3.5
|
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 2 hours; 20 minute heat shock at 65C
3. Column Purification, elute with 30 uL EB
4. Measure concentrations in nanodrop
Nanodrop Results[edit]
Sample
|
V6S1AB
|
V6S1CD
|
V6S2AB
|
V6S2CD
|
ng/uL
|
128.6
|
125.8
|
147.2
|
150
|
ug in 30 uL
|
3.9
|
3.8
|
4.4
|
4.5
|
total
|
|
7.6
|
|
8.9
|
TBE Gel[edit]
1. Lanes; (5.5 uL TBE; 2 uL 6x buffer, 0.5 uL sample; load 7.2 uL for all)
Lane
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
10
|
11
|
12
|
Sample
|
|
|
|
10bp ladder
|
V6S1AB
|
V6S1CD
|
|
V6S2AB
|
V6S2CD
|
|
|
|
2. Run for 25 minutes at 220V
3. Stain with 3 uL SYBR gold for 2 minutes
Gel Image
File:2013-11-14-DpnII.jpg