AlanFung:LabNotes/Capturing/2012-8-10: Difference between revisions

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*Add 2 drops of mineral oil to prevent evaporation
*Add 2 drops of mineral oil to prevent evaporation
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|- style="background-color:#BFBFBF;font-size:11pt;font-weight:bold" align="center"
|- style="background-color:#BFBFBF;font-size:8pt;font-weight:bold" align="center"
|style="text-decoration:underline" width="270" height="13" | ChIPK36me3
|style="text-decoration:underline" width="270" height="13" | ChIPK36me3
| width="121" | Concentration ng/ul
| width="121" | Concentration ng/ul
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| width="33" | Total
| width="33" | Total


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
|style="text-decoration:none" height="13" | GM12878
|style="text-decoration:none" height="13" | GM12878
| align="center" | 0.90
| align="center" | 0.90
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | P10
| height="13" | P10
| align="center" | 1.20
| align="center" | 1.20
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | DF2
| height="13" | DF2
| align="center" | 0.70
| align="center" | 0.70
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H0
| height="13" | H0
| align="center" | 1.00
| align="center" | 1.00
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H7
| height="13" | H7
| align="center" | 0.60
| align="center" | 0.60
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H14
| height="13" | H14
| align="center" | 0.50
| align="center" | 0.50
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H16
| height="13" | H16
| align="center" | 0.60
| align="center" | 0.60
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" |    
| height="13" |    
  |    
  |    
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| align="center" | &nbsp;
| align="center" | &nbsp;


|- style="background-color:#BFBFBF;font-size:11pt;font-weight:bold" align="center"
|- style="background-color:#BFBFBF;font-size:8pt;font-weight:bold" align="center"
|style="text-decoration:underline" height="13" | ChIPK27me3
|style="text-decoration:underline" height="13" | ChIPK27me3
  | Concentration ng/ul
  | Concentration ng/ul
Line 92: Line 92:
  | Total
  | Total


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
|style="text-decoration:none" height="13" | GM12878
|style="text-decoration:none" height="13" | GM12878
| align="center" | 1.20
| align="center" | 1.20
Line 101: Line 101:
| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | GM12878 Round 2
| height="13" | GM12878 Round 2
| align="center" | 2.86
| align="center" | 2.86
Line 110: Line 110:
| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="12" | P10
| height="12" | P10
| align="center" | 1.30
| align="center" | 1.30
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | DF2
| height="13" | DF2
| align="center" | 2
| align="center" | 2
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H0
| height="13" | H0
| align="center" | 2.4
| align="center" | 2.4
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H7
| height="13" | H7
| align="center" | 1.2
| align="center" | 1.2
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H14
| height="13" | H14
| align="center" | 0.5
| align="center" | 0.5
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H16
| height="13" | H16
| align="center" | 1.3
| align="center" | 1.3
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" |    
| height="13" |    
  |    
  |    
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| align="center" | &nbsp;
| align="center" | &nbsp;


|- style="background-color:#BFBFBF;font-size:11pt;font-weight:bold" align="center"
|- style="background-color:#BFBFBF;font-size:8pt;font-weight:bold" align="center"
|style="text-decoration:underline" height="13" | WCE
|style="text-decoration:underline" height="13" | WCE
  | Concentration ng/ul
  | Concentration ng/ul
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  | Total
  | Total


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
|style="text-decoration:none" height="13" | GM12878
|style="text-decoration:none" height="13" | GM12878
| align="center" | 35.39
| align="center" | 35.39
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| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | GM12878 Round 2
| height="13" | GM12878 Round 2
| align="center" | 2.86
| align="center" | 2.86
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | P10
| height="13" | P10
| align="center" | 35.39
| align="center" | 35.39
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| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | P10 Round 2
| height="13" | P10 Round 2
| align="center" | 2.86
| align="center" | 2.86
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | DF2
| height="13" | DF2
| align="center" | 35.39
| align="center" | 35.39
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| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | DF2 Round 2
| height="13" | DF2 Round 2
| align="center" | 2.86
| align="center" | 2.86
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| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H0
| height="13" | H0
| align="center" | 35.39
| align="center" | 35.39
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| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H7
| height="13" | H7
| align="center" | 35.39
| align="center" | 35.39
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| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H14
| height="13" | H14
| align="center" | 35.39
| align="center" | 35.39
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| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H16
| height="13" | H16
| align="center" | 35.39
| align="center" | 35.39
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| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" |    
| height="13" |    
  |    
  |    
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| align="center" | &nbsp;
| align="center" | &nbsp;


|- style="background-color:#BFBFBF;font-size:11pt;font-weight:bold" align="center"
|- style="background-color:#BFBFBF;font-size:8pt;font-weight:bold" align="center"
|style="text-decoration:underline" height="13" | cDNA
|style="text-decoration:underline" height="13" | cDNA
  | Concentration ng/ul
  | Concentration ng/ul
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  | Total
  | Total


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
|style="text-decoration:none" height="13" | GM
|style="text-decoration:none" height="13" | GM
| align="center" | 38.12
| align="center" | 38.12
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| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H0
| height="13" | H0
| align="center" | 38.12
| align="center" | 38.12
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| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H7
| height="13" | H7
| align="center" | 38.12
| align="center" | 38.12
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| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H14
| height="13" | H14
| align="center" | 38.12
| align="center" | 38.12
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| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H16
| height="13" | H16
| align="center" | 38.12
| align="center" | 38.12
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| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" |    
| height="13" |    
  |    
  |    
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| align="center" | &nbsp;
| align="center" | &nbsp;


|- style="background-color:#BFBFBF;font-size:11pt;font-weight:bold" align="center"
|- style="background-color:#BFBFBF;font-size:8pt;font-weight:bold" align="center"
|style="text-decoration:underline" height="13" | gDNA
|style="text-decoration:underline" height="13" | gDNA
  | Concentration ng/ul
  | Concentration ng/ul
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  | Total
  | Total


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
|style="text-decoration:none" height="13" | H7
|style="text-decoration:none" height="13" | H7
| align="center" | 38.12
| align="center" | 38.12
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| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H14
| height="13" | H14
| align="center" | 38.12
| align="center" | 38.12
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| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | H16
| height="13" | H16
| align="center" | 38.12
| align="center" | 38.12

Revision as of 00:40, 11 August 2012

Capturing Remaining Gimelbrant lab Sample

  • Add 2 drops of mineral oil to prevent evaporation
ChIPK36me3 Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
GM12878 0.90 40.00 0.45 2 7.6 50
P10 1.20 40.00 0.60 2 7.4 50
DF2 0.70 40.00 0.35 2 7.7 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
GM12878 Round 2 2.86 35.00 1.25 2 11.8 50
P10 1.30 40.00 0.65 2 7.4 50
DF2 2 40.00 1.00 2 7.0 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
GM12878 Round 2 2.86 35.00 1.25 2 11.8 50
P10 35.39 5.65 2.49 2 9.9 20
P10 Round 2 2.86 35.00 1.25 2 11.8 50
DF2 35.39 5.65 2.49 2 9.9 20
DF2 Round 2 2.86 35.00 1.25 2 11.8 50
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
  • Make sure we have enough reagent before start,
  • need 50ul probes, check
  • need 66ul 10X Ampligase Buffer,
  • need 33ul dNTP (10uM),
  • need 13.2ul dNTP (10uM)
  • need 13.2ul Stoffel (10U/ul)
  • need 66ul Exo I (20U/ul)
  • need 66ul ExoIII (200U/ul)

Prepare SLN Mix

  • Dilute 1mM dNTP 1 in 100 to 10uM dNTP
SLN Mix for 20ul Rxn 1x 16.5x
dNTP (10uM) 0.5 8.25
Stoffel (10U/ul) 0.2 3.30
Ampligase (5U/ul) 0.2 3.30
H2O 0.1 1.65
   
SLN Mix for 50ul Rxn 1x 19.8x
dNTP (10uM) 1.25 24.75
Stoffel (10U/ul) 0.5 9.90
Ampligase (5U/ul) 0.5 9.90
H2O 0.25 4.95

Prepare Exo I and III Mix

Exo I & III for 20ul Rxn 1X 16.5x
Exo I (20U/ul) 1 16.5
ExoIII (200U/ul) 1 16.5
     
Exo I & III for 50ul Rxn 1X 19.8x
Exo I (20U/ul) 2.5 49.5
ExoIII (200U/ul) 2.5 49.5
  • When adding regents to the tube make sure you penetrate the oil layer
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