Daniel:Notebook/HiResChrPaint/2013-3-26: Difference between revisions
Jump to navigation
Jump to search
>Djacobse No edit summary |
>Djacobse No edit summary |
||
(6 intermediate revisions by the same user not shown) | |||
Line 1: | Line 1: | ||
=Probe Set Prep (Started 3/26/2013)= | =Probe Set Prep (Started 3/26/2013)= | ||
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | |||
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 and Ethanol Precipitation== | ||
Line 78: | Line 83: | ||
|style="font-weight:bold" | V6S2 | |style="font-weight:bold" | V6S2 | ||
|style="font-weight:bold" | V6S3 | |style="font-weight:bold" | V6S3 | ||
|style="font-weight:bold" | | |style="font-weight:bold" | V6S4 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
Line 139: | Line 144: | ||
2. qPCR | 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 ([[Daniel:Notebook/HiResChrPaint/2013-3-27|Continued on 3/27/2103]]) | |||
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]] |
Latest revision as of 00:07, 21 June 2013
Probe Set Prep (Started 3/26/2013)[edit]
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[edit]
Expansion PCR[edit]
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 | V6S4 | |
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[edit]
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)