Daniel:Notebook/HiResChrPaint/2013-3-26: Difference between revisions

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=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==
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|style="font-weight:bold" | V6S2
|style="font-weight:bold" | V6S2
|style="font-weight:bold" | V6S3
|style="font-weight:bold" | V6S3
|style="font-weight:bold" | V6S6
|style="font-weight:bold" | V6S4


|- style="font-size:12pt" align="center"
|- style="font-size:12pt" align="center"
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   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]

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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)

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