AlanFung:LabNotes/Capturing/2012-8-10

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Revision as of 01:48, 15 August 2012 by >Alan6017518 (→‎Indexing qPCR)
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Capturing Remaining Gimelbrant lab Sample

  • Add 2 drops of mineral oil to prevent evaporation
  • Samples stored at tall -20C Alan Rack white box red label "Gimelbrant Lab"
ChIPK36me3 Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
GM12878 0.90 40.00 0.45 2 7.6 50
H0 1.00 40.00 0.50 2 7.5 50
H7 0.60 40.00 0.30 2 7.7 50
H14 0.50 40.00 0.25 2 7.8 50
H16 0.60 40.00 0.30 2 7.7 50
                
ChIPK27me3 Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
GM12878 1.20 40.00 0.60 2 7.4 50
H0 2.4 40.00 1.20 2 6.8 50
H7 1.2 40.00 0.60 2 7.4 50
H14 0.5 40.00 0.25 2 7.8 50
H16 1.3 40.00 0.65 2 7.4 50
                
WCE Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
GM12878 35.39 5.65 2.49 2 9.9 20
P10 35.39 5.65 2.49 2 9.9 20
DF2 35.39 5.65 2.49 2 9.9 20
H0 35.39 5.65 2.49 2 9.9 20
H7 35.39 5.65 2.49 2 9.9 20
H14 35.39 5.65 2.49 2 9.9 20
H16 35.39 5.65 2.49 2 9.9 20
                
cDNA Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
GM 38.12 5.25 2.49 2 10.3 20
H0 38.12 5.25 2.49 2 10.3 20
H7 38.12 5.25 2.49 2 10.3 20
H14 38.12 5.25 2.49 2 10.3 20
H16 38.12 5.25 2.49 2 10.3 20
                
gDNA Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
H7 38.12 5.25 2.49 2 10.3 20
H14 38.12 5.25 2.49 2 10.3 20
H16 38.12 5.25 2.49 2 10.3 20
  • When adding regents to the tube make sure you penetrate the oil layer
Store under βCES36K
95C 2min -> -0.2C/sec to 60C -> 60C 24h 
   -> add 1ul/2.5ul(for 50ul rxn) SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul) 
   -> 60C 18h -> 94C 1min -> 37c 1min -> add 2ul(5ul for 50ul rxn) Exo I&III
   -> 37C 2h -> 90C 5min -> 4C hold

Prepare SLN Mix

  • Dilute 1mM dNTP 1 in 100 to 10uM dNTP
SLN Mix for 20ul Rxn 1x 16x
dNTP (10uM) 0.5 8
Stoffel (10U/ul) 0.2 3.2
Ampligase (5U/ul) 0.2 3.2
H2O 0.1 1.6
     
SLN Mix for 50ul Rxn 1x 11x
dNTP (10uM) 1.25 13.75
Stoffel (10U/ul) 0.5 5.5
Ampligase (5U/ul) 0.5 5.5
H2O 0.25 2.75

Prepare Exo I and Exo III Mix

Exo I & III for 20ul Rxn 1X 16x
Exo I (20U/ul) 1 16
ExoIII (200U/ul) 1 16
     
Exo I & III for 50ul Rxn 1X 11x
Exo I (20U/ul) 2.5 6.25
ExoIII (200U/ul) 2.5 6.25

Indexing qPCR

  • Perform one step qPCR using Indexing seq. primer