Daniel:Notebook/HiResChrPaint/2013-3-26
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Probe Set Prep (Started 3/26/2013)
Purpose: To generate a probe set for hi-res chromosome painting. Two groups are amplified in this protocol, the V4 group (sets V4S1-4) and the V6 group(V6S1-4).
Expansion PCR and Ethanol Precipitation
Expansion PCR
1. Set up 8 reactions for each of the 8 probes (64 reactions total)
per rxn | V4S1 | V4S2 | V4S3 | V4S4 | |
20 nM OligoPool | 0.2 | 1.7 | 1.7 | 1.7 | 1.7 |
2X Kapa SYBR qPCR MM | 50 | 425 | 425 | 425 | 425 |
100 uM AP1V41U | 0.4 | 3.4 | 3.4 | 3.4 | 3.4 |
100 uM AP2V4 | 0.4 | 3.4 | 3.4 | 3.4 | 3.4 |
2 mM aa-dUTP | 3 | 25.5 | 25.5 | 25.5 | 25.5 |
H20 | 46 | 391 | 391 | 391 | 391 |
Total Volume | 100 | 850 | 850 | 850 | 850 |
per rxn | V6S1 | V6S2 | V6S3 | V6S6 | |
20 nM OligoPool | 0.2 | 1.7 | 1.7 | 1.7 | 1.7 |
2X Kapa SYBR qPCR MM | 50 | 425 | 425 | 425 | 425 |
100 uM AP1V61U | 0.4 | 3.4 | 3.4 | 3.4 | 3.4 |
100 uM AP2V6 | 0.4 | 3.4 | 3.4 | 3.4 | 3.4 |
2 mM aa-dUTP | 3 | 25.5 | 25.5 | 25.5 | 25.5 |
H20 | 46 | 391 | 391 | 391 | 391 |
Total Volume | 100 | 850 | 850 | 850 | 850 |
2. qPCR 95 C 30 seconds Repeat 16x 95 C 3 seconds 55 C 30 seconds 72 C 20 seconds Read Plate 72 C 2 min 15 C Hold stopped qPCR for all samples except V4S1 and V4S2 at cycle 12 stopped qPCR for V4S1 and V4S2 at final cycle
Ethanol Precipitation
1. Pool all samples for given probe set into 15 mL eppendorf tube (8 tubes, 800 uL each) 2. Add: 2000 uL EtOH (Rec 2.5X) 80 uL NaOAc (Rec 0.1X) 2.7 uL Glycoblue (Rec 1/300X) 3. Freeze tube overnight at -80C (Continued on 3/27/2103)