Revision as of 01:22, 9 August 2013 by >Djacobse
S2 Probe Prep (ULS)
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I have some more Alexa 488 from the ULYSIS kits left, and they recommend using it within a month. Since it has been almost a month since I opened the bottle, I guess I should make more (once coupled the dye is stable for several more months). Besides, I could use the ULS labeled probe as a comparison for future FISH experiments.
qPCR Amplification
1. Set up master reactions according to table
|
20 nM OligoPool
|
2X Kapa SYBR qPCR MM
|
100 uM AP1V41U
|
100 uM AP2V4
|
100 uM AP1V61U
|
100 uM AP2V6
|
50 mM aa-dUTP
|
H20
|
Total Volume
|
per rxn
|
0.2
|
50
|
0.4
|
0.4
|
0.4
|
0.4
|
0
|
48.2
|
100
|
V4S2 (12.5x)
|
2.5
|
625
|
5
|
5
|
0
|
0
|
0
|
612.5
|
1250
|
V6S2 (12.5x)
|
2.5
|
625
|
0
|
0
|
5
|
5
|
0
|
612.5
|
1250
|
2. Aliquot 100 uL into each of 12 qPCR tubes (24 in total for both reactions)
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. Pool 3 samples together each (300 uL each, 4 pools per set, 8 total)
5. Column puficiation, elute with 40 uL EB
6. Measure in Nanodrop
qPCR Results
Nanodrop Results
Sample
|
V4S2A
|
V4S2B
|
V4S2C
|
V4S2D
|
V6S2A
|
V6S2B
|
V6S2C
|
V6S2D
|
ng/uL
|
64.7
|
62.6
|
69.1
|
79.2
|
139
|
126.8
|
127.3
|
127.2
|
ug in 40 uL
|
2.6
|
2.5
|
2.8
|
3.2
|
5.6
|
5.1
|
5.1
|
5.1
|
Total ug
|
|
|
|
11.0
|
|
|
|
20.8
|
DpnII Digestion
1. Prepare Samples
Sample
|
V4S2A
|
V4S2B
|
V4S2C
|
V4S2D
|
V6S2A
|
V6S2B
|
V6S2C
|
V6S2D
|
ng/uL
|
64.7
|
62.6
|
69.1
|
79.2
|
139
|
126.8
|
127.3
|
127.2
|
ug in 35 uL
|
2.3
|
2.2
|
2.4
|
2.8
|
4.9
|
4.4
|
4.5
|
4.5
|
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 8/9/2013
3. Column purification, elute with 30 uL EB
4. Measure in Nanodrop