Revision as of 23:36, 31 May 2013 by >Djacobse
Probe Prep Test 3[edit]
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Purpose: With the relative success of previous results I am now ready to amplify another probe set. At the same time
I want to test the other dye coupling method (ULYSIS), which non-specifically couples with G residues. I will have Alan order the ULYSIS dye and
will therefore expand aa-dUTP coupled probes and normal probes. I suspect the normal probes will give me better yields throughout the process.
Expansion qPCR[edit]
1. Set up master reactions according to table
aa-dUTP coupled probes[edit]
|
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
|
0.1
|
46
|
100
|
V4S2 (12.5x)
|
2.5
|
625
|
5
|
5
|
0
|
0
|
1.5
|
611
|
1250
|
V6S2 (12.5x)
|
2.5
|
625
|
0
|
0
|
5
|
5
|
1.5
|
611
|
1250
|
Normal Probes (no aa-dUTP)[edit]
|
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. 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 overnight (continued 6/1/2013)