Daniel:Notebook/HiResChrPaint/2013-5-31

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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)