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

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==Capturing Remaining Gimelbrant lab Sample==
==Capturing Remaining Gimelbrant lab Sample==
*Add 2 drops of mineral oil to prevent evaporation
*Add 2 drops of mineral oil to prevent evaporation
*Samples stored at tall -20C Alan Rack white box red label "Gimelbrant Lab"
{| 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
Line 11: Line 12:
| 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
Line 20: Line 21:
| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | P10
| align="center" | 1.20
| align="center" | 40.00
| align="center" | 0.60
| align="center" | 2
| align="center" | 7.4
| align="center" | 50
 
|- style="font-size:11pt" align="center"
| height="13" | DF2
| align="center" | 0.70
| align="center" | 40.00
| align="center" | 0.35
| align="center" | 2
| align="center" | 7.7
| align="center" | 50
 
|- style="font-size:11pt" align="center"
| height="13" | H0
| height="13" | H0
| align="center" | 1.00
| align="center" | 1.00
Line 47: Line 30:
| 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
Line 56: Line 39:
| 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
Line 65: Line 48:
| 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
Line 74: Line 57:
| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" |    
| height="13" |    
  |    
  |    
Line 83: Line 66:
| 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 75:
  | 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 84:
| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | GM12878 Round 2
| align="center" | 2.86
| align="center" | 35.00
| align="center" | 1.25
| align="center" | 2
| align="center" | 11.8
| align="center" | 50
 
|- style="font-size:11pt" align="center"
| height="12" | P10
| align="center" | 1.30
| align="center" | 40.00
| align="center" | 0.65
| align="center" | 2
| align="center" | 7.4
| align="center" | 50
 
|- style="font-size:11pt" align="center"
| height="13" | DF2
| align="center" | 2
| align="center" | 40.00
| align="center" | 1.00
| align="center" | 2
| align="center" | 7.0
| align="center" | 50
 
|- style="font-size:11pt" align="center"
| height="13" | H0
| height="13" | H0
| align="center" | 2.4
| align="center" | 2.4
Line 137: Line 93:
| 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
Line 146: Line 102:
| 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
Line 155: Line 111:
| 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
Line 164: Line 120:
| align="center" | 50
| align="center" | 50


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" |    
| height="13" |    
  |    
  |    
Line 173: Line 129:
| 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
Line 182: Line 138:
  | 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
Line 191: Line 147:
| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | GM12878 Round 2
| align="center" | 2.86
| align="center" | 35.00
| align="center" | 1.25
| align="center" | 2
| align="center" | 11.8
| align="center" | 50
 
|- style="font-size:11pt" align="center"
| height="13" | P10
| height="13" | P10
| align="center" | 35.39
| align="center" | 35.39
Line 209: Line 156:
| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | P10 Round 2
| align="center" | 2.86
| align="center" | 35.00
| align="center" | 1.25
| align="center" | 2
| align="center" | 11.8
| align="center" | 50
 
|- style="font-size:11pt" align="center"
| height="13" | DF2
| height="13" | DF2
| align="center" | 35.39
| align="center" | 35.39
Line 227: Line 165:
| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | DF2 Round 2
| align="center" | 2.86
| align="center" | 35.00
| align="center" | 1.25
| align="center" | 2
| align="center" | 11.8
| align="center" | 50
 
|- style="font-size:11pt" align="center"
| height="13" | H0
| height="13" | H0
| align="center" | 35.39
| align="center" | 35.39
Line 245: Line 174:
| 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
Line 254: Line 183:
| 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
Line 263: Line 192:
| 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
Line 272: Line 201:
| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" |    
| height="13" |    
  |    
  |    
Line 281: Line 210:
| 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
Line 290: Line 219:
  | 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
Line 299: Line 228:
| 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
Line 308: Line 237:
| 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
Line 317: Line 246:
| 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
Line 326: Line 255:
| 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
Line 335: Line 264:
| align="center" | 20
| align="center" | 20


|- style="font-size:11pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" |    
| height="13" |    
  |    
  |    
Line 344: Line 273:
| 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
Line 353: Line 282:
  | 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
Line 362: Line 291:
| 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
Line 371: Line 300:
| 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
Line 381: Line 310:


|}
|}
*Make sure we have enough reagent before start,
*When adding regents to the tube make sure you penetrate the oil layer
*need 50ul probes, check
Store under βCES36K
*need 66ul 10X Ampligase Buffer,
95C 2min -> -0.2C/sec to 60C -> 60C 24h
*need 33ul dNTP (10uM),
    -> add 1ul/2.5ul(for 50ul rxn) SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul)  
*need 13.2ul dNTP (10uM)
    -> 60C 18h -> 94C 1min -> 37c 1min -> add 2ul(5ul for 50ul rxn) Exo I&III
*need 13.2ul Stoffel (10U/ul)
    -> 37C 2h -> 90C 5min -> 4C hold
*need 66ul Exo I (20U/ul)
*need 66ul ExoIII (200U/ul)
===Prepare SLN Mix===
===Prepare SLN Mix===
*Dilute 1mM dNTP 1 in 100 to 10uM dNTP
*Dilute 1mM dNTP 1 in 100 to 10uM dNTP
{| 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:#B1A0C7;font-size:8pt" align="center" valign="bottom"
|- style="font-size:8pt;font-weight:bold" align="center"
|style="font-weight:bold;text-decoration:underline" width="270" height="13" | SLN Mix for 20ul Rxn
|style="text-decoration:underline" width="81" height="15" | SLN Mix for 20ul Rxn
|style="font-weight:bold;text-decoration:none" width="121" | 1x
|style="text-decoration:none" width="112" | 1x
| width="41" | 16.5x
| width="83" | 16x


|- style="font-size:8pt" align="center" valign="bottom"
|- style="font-size:8pt" align="center"
|style="font-weight:bold" height="13" | dNTP (10uM)
|style="font-weight:bold" height="15" | dNTP (10uM)
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 8.25
| align="center" | 8


|- style="font-size:8pt" align="center" valign="bottom"
|- style="font-size:8pt" align="center"
|style="font-weight:bold" height="13" | Stoffel (10U/ul)
|style="font-weight:bold" height="15" | Stoffel (10U/ul)
| align="center" | 0.2
| align="center" | 0.2
| align="center" | 3.30
| align="center" | 3.2


|- style="font-size:8pt" align="center" valign="bottom"
|- style="font-size:8pt" align="center"
|style="font-weight:bold" height="13" | Ampligase (5U/ul)
|style="font-weight:bold" height="15" | Ampligase (5U/ul)
| align="center" | 0.2
| align="center" | 0.2
| align="center" | 3.30
| align="center" | 3.2


|- style="font-size:8pt" align="center" valign="bottom"
|- style="font-size:8pt" align="center"
|style="font-weight:bold" height="13" | H2O
|style="font-weight:bold" height="15" | H2O
| align="center" | 0.1
| align="center" | 0.1
| align="center" | 1.65
| align="center" | 1.6


|- style="font-size:8pt" align="center" valign="bottom"
|- style="font-size:8pt" align="center"
| align="center" height="13" | &nbsp;
| align="center" height="15" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" |


|- style="background-color:#B1A0C7;font-size:8pt" align="center" valign="bottom"
|- style="font-size:8pt;font-weight:bold" align="center"
|style="font-weight:bold;text-decoration:underline" height="13" | SLN Mix for 50ul Rxn
|style="text-decoration:underline" height="15" | SLN Mix for 50ul Rxn
|style="font-weight:bold;text-decoration:none" | 1x
|style="text-decoration:none" | 1x
  | 19.8x
  | 11x


|- style="font-size:8pt" align="center" valign="bottom"
|- style="font-size:8pt" align="center"
|style="font-weight:bold" height="13" | dNTP (10uM)
|style="font-weight:bold" height="15" | dNTP (10uM)
| align="center" | 1.25
| align="center" | 1.25
| align="center" | 24.75
| align="center" | 13.75


|- style="font-size:8pt" align="center" valign="bottom"
|- style="font-size:8pt" align="center"
|style="font-weight:bold" height="13" | Stoffel (10U/ul)
|style="font-weight:bold" height="15" | Stoffel (10U/ul)
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 9.90
| align="center" | 5.5


|- style="font-size:8pt" align="center" valign="bottom"
|- style="font-size:8pt" align="center"
|style="font-weight:bold" height="13" | Ampligase (5U/ul)
|style="font-weight:bold" height="15" | Ampligase (5U/ul)
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 9.90
| align="center" | 5.5


|- style="font-size:8pt" align="center" valign="bottom"
|- style="font-size:8pt" align="center"
|style="font-weight:bold" height="13" | H2O
|style="font-weight:bold" height="15" | H2O
| align="center" | 0.25
| align="center" | 0.25
| align="center" | 4.95
| align="center" | 2.75


|}
|}
===Prepare Exo I and III Mix===
===Prepare Exo I and Exo III Mix===
{| 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:#538DD5;font-size:8pt" align="center"
|- style="background-color:#FFFF00;font-size:8pt;font-weight:bold" align="center"
|style="font-weight:bold;text-decoration:underline" width="121" height="13" | Exo I & III for 20ul Rxn
|style="text-decoration:underline" width="81" height="15" | Exo I & III for 20ul Rxn
|style="font-weight:bold;text-decoration:none" width="39" | 1X
|style="text-decoration:none" width="112" | 1X
| width="33" | 16.5x
| width="83" | 16x


|- style="font-size:8pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | Exo I (20U/ul)
| height="15" | Exo I (20U/ul)
| align="center" | 1
| align="center" | 1
| align="center" | 16.5
| align="center" | 16


|- style="font-size:8pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | ExoIII (200U/ul)
| height="15" | ExoIII (200U/ul)
| align="center" | 1
| align="center" | 1
| align="center" | 16.5
| align="center" | 16


|- style="font-size:8pt" align="center"
|- style="font-size:8pt" align="center"
| align="center" height="13" | &nbsp;
| align="center" height="15" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;


|- style="background-color:#538DD5;font-size:8pt" align="center"
|- style="background-color:#FFFF00;font-size:8pt;font-weight:bold" align="center"
|style="font-weight:bold;text-decoration:underline" height="13" | Exo I & III for 50ul Rxn
|style="text-decoration:underline" height="15" | Exo I & III for 50ul Rxn
|style="font-weight:bold;text-decoration:none" | 1X
|style="text-decoration:none" | 1X
  | 19.8x
  | 11x


|- style="font-size:8pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | Exo I (20U/ul)
| height="15" | Exo I (20U/ul)
| align="center" | 2.5
| align="center" | 2.5
| align="center" | 49.5
| align="center" | 6.25


|- style="font-size:8pt" align="center"
|- style="font-size:8pt" align="center"
| height="13" | ExoIII (200U/ul)
| height="15" | ExoIII (200U/ul)
| align="center" | 2.5
| align="center" | 2.5
| align="center" | 49.5
| align="center" | 6.25
 
|}
==Indexing qPCR==
*Perform one step qPCR using Indexing seq. primer
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|- style="font-size:8pt" align="center"
|style="background-color:#FFFF00;font-weight:bold;text-decoration:underline" width="126" height="11" | Content (50ul Capture) 4 rxn
|style="background-color:#FFFF00;font-weight:bold" width="35" | Volume
|style="background-color:#FFFF00;font-weight:bold" width="70" | Master Mix
|style="background-color:#FFFF00;font-weight:bold" width="81" | ChIPK36me3
|style="background-color:#FFFF00;font-weight:bold" width="65" | Index
|style="background-color:#FFFF00;font-weight:bold" width="75" | ChIPK27me3
|style="background-color:#FFFF00;font-weight:bold" width="65" | Index
|style="text-decoration:none" align="center" width="65" | &nbsp;
| align="center" width="65" | &nbsp;
 
|- style="font-size:8pt" align="center"
| height="11" | Captured DNA
| align="center" | 27
| Do not add to MM
| GM12878
| align="center" | 1
| GM12878
| align="center" | 10
| align="center" | &nbsp;
| align="center" | &nbsp;
 
|- style="font-size:8pt" align="center"
| height="11" | 2X Kapa HiFi mix
| align="center" | 50
| align="center" | 550
| H0
| align="center" | 2
| H0
| align="center" | 11
| align="center" | &nbsp;
| align="center" | &nbsp;
 
|- style="font-size:8pt" align="center"
| height="11" | 10uM AmpF6.3Sol
| align="center" | 4
| align="center" | 44
| H7
| align="center" | 3
| H7
| align="center" | 12
| align="center" | &nbsp;
| align="center" | &nbsp;
 
|- style="font-size:8pt" align="center"
| height="11" | 10uM AmpR6.3Ind
| align="center" | 4
| Do not add to MM
| H14
| align="center" | 4
| H14
| align="center" | 13
| align="center" | &nbsp;
| align="center" | &nbsp;
 
|- style="font-size:8pt" align="center"
| height="11" | H2O
| align="center" | 15
| align="center" | 165
| H16
| align="center" | 9
| H16
| align="center" | 14
| align="center" | &nbsp;
| align="center" | &nbsp;
 
|- style="font-size:8pt" align="center"
| height="11" | SYBR Green 50X
| align="center" | 0.4
| align="center" | 4.4
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
 
|- style="font-size:8pt" align="center"
| align="center" height="11" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
 
|- style="background-color:#3366FF;font-size:8pt;font-weight:bold" align="center"
|style="text-decoration:underline" height="11" | Content (20ul Capture) 7rxn
| Volume
| Master Mix
| WCE
| Index
| cDNA
| Index
| gDNA
| Index
 
|- style="font-size:8pt" align="center"
|style="text-decoration:none" height="11" | Captured DNA
| align="center" | 11
| Do not add to MM
| GM12878
| align="center" | 15
| GM
| align="center" | 22
| H7
| align="center" | 27
 
|- style="font-size:8pt" align="center"
| height="11" | 2X Kapa SYBG qPCR master mix
| align="center" | 50
| align="center" | 825
| P10
| align="center" | 16
| H0
| align="center" | 23
| H14
| align="center" | 28
 
|- style="font-size:8pt" align="center"
| height="11" | 10uM AmpF6.3Sol
| align="center" | 4
| align="center" | 66
| DF2
| align="center" | 17
| H7
| align="center" | 24
| H16
| align="center" | 29
 
|- style="font-size:8pt" align="center"
| height="11" | 10uM AmpR6.3Ind
| align="center" | 4
| Do not add to MM
| H0
| align="center" | 18
| H14
| align="center" | 25
| align="center" | &nbsp;
| align="center" | &nbsp;
 
|- style="font-size:8pt" align="center"
| height="11" | H2O
| align="center" | 31
| align="center" | 511.5
| H7
| align="center" | 19
| H16
| align="center" | 26
| align="center" | &nbsp;
| align="center" | &nbsp;
 
|- style="font-size:8pt" align="center"
| height="11" | SYBR Green 50X
| align="center" | 0.4
| align="center" | 6.6
| H14
| align="center" | 20
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
 
|- style="font-size:8pt" align="center"
| align="center" height="11" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| H16
| align="center" | 21
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;


|}
|}
*When adding regents to the tube make sure you penetrate the oil layer
  95C 2min -> (98C 20S -> 58C 20S -> 72C 20S) x 6 -> (98C 10S -> 72C 20S) x40
  95C 2min -> -0.2C/sec to 60C -> 60C 24h
Monitor the reactions and terminate the program right before the amplification curves reach the plateau.
    -> add 1ul/2.5ul(for 50ul rxn) SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul)  
==Indexing PCR for 3 samples==
    -> 60C 18h -> 94C 1min -> 37c 1min -> add 2ul(5ul for 50ul rxn) Exo I&III
    -> 37C 2h -> 90C 5min -> 4C hold

Latest revision as of 13:41, 18 August 2012

Capturing Remaining Gimelbrant lab Sample[edit]

  • 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[edit]

  • 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[edit]

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[edit]

  • Perform one step qPCR using Indexing seq. primer
Content (50ul Capture) 4 rxn Volume Master Mix ChIPK36me3 Index ChIPK27me3 Index    
Captured DNA 27 Do not add to MM GM12878 1 GM12878 10    
2X Kapa HiFi mix 50 550 H0 2 H0 11    
10uM AmpF6.3Sol 4 44 H7 3 H7 12    
10uM AmpR6.3Ind 4 Do not add to MM H14 4 H14 13    
H2O 15 165 H16 9 H16 14    
SYBR Green 50X 0.4 4.4            
                 
Content (20ul Capture) 7rxn Volume Master Mix WCE Index cDNA Index gDNA Index
Captured DNA 11 Do not add to MM GM12878 15 GM 22 H7 27
2X Kapa SYBG qPCR master mix 50 825 P10 16 H0 23 H14 28
10uM AmpF6.3Sol 4 66 DF2 17 H7 24 H16 29
10uM AmpR6.3Ind 4 Do not add to MM H0 18 H14 25    
H2O 31 511.5 H7 19 H16 26    
SYBR Green 50X 0.4 6.6 H14 20        
      H16 21        
95C 2min -> (98C 20S -> 58C 20S -> 72C 20S) x 6 -> (98C 10S -> 72C 20S) x40
Monitor the reactions and terminate the program right before the amplification curves reach the plateau.

Indexing PCR for 3 samples[edit]