Daniel:Notebook/HiResChrPaint/2013-5-31
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Probe Prep Test 3[edit]
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)