AlanFung:LabNotes/Capturing/2012-8-10: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 No edit summary |
||
(40 intermediate revisions by the same user not shown) | |||
Line 1: | Line 1: | ||
==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: | |- 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: | |- 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: | |- style="font-size:8pt" 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: | |- 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: | |- 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: | |- 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: | |- style="font-size:8pt" align="center" | ||
| height="13" | | | height="13" | | ||
| | | | ||
Line 83: | Line 66: | ||
| align="center" | | | align="center" | | ||
|- style="background-color:#BFBFBF;font-size: | |- 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: | |- 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: | |- style="font-size:8pt" 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: | |- 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: | |- 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: | |- 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: | |- style="font-size:8pt" align="center" | ||
| height="13" | | | height="13" | | ||
| | | | ||
Line 173: | Line 129: | ||
| align="center" | | | align="center" | | ||
|- style="background-color:#BFBFBF;font-size: | |- 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: | |- 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: | |- style="font-size:8pt" 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: | |- style="font-size:8pt" 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: | |- style="font-size:8pt" 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: | |- 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: | |- 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: | |- 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: | |- style="font-size:8pt" align="center" | ||
| height="13" | | | height="13" | | ||
| | | | ||
Line 281: | Line 210: | ||
| align="center" | | | align="center" | | ||
|- style="background-color:#BFBFBF;font-size: | |- 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: | |- 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: | |- 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: | |- 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: | |- 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: | |- 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: | |- style="font-size:8pt" align="center" | ||
| height="13" | | | height="13" | | ||
| | | | ||
Line 344: | Line 273: | ||
| align="center" | | | align="center" | | ||
|- style="background-color:#BFBFBF;font-size: | |- 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: | |- 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: | |- 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: | |- style="font-size:8pt" align="center" | ||
| height="13" | H16 | | height="13" | H16 | ||
| align="center" | 38.12 | | align="center" | 38.12 | ||
Line 381: | Line 310: | ||
|} | |} | ||
* | *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=== | ===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=" | |- style="font-size:8pt;font-weight:bold" align="center" | ||
|style=" | |style="text-decoration:underline" width="81" height="15" | SLN Mix for 20ul Rxn | ||
|style=" | |style="text-decoration:none" width="112" | 1x | ||
| width=" | | width="83" | 16x | ||
|- style="font-size:8pt" align="center | |- style="font-size:8pt" align="center" | ||
|style="font-weight:bold" height=" | |style="font-weight:bold" height="15" | dNTP (10uM) | ||
| align="center" | 0.5 | | align="center" | 0.5 | ||
| align="center" | 8 | | align="center" | 8 | ||
|- style="font-size:8pt" align="center | |- style="font-size:8pt" align="center" | ||
|style="font-weight:bold" height=" | |style="font-weight:bold" height="15" | Stoffel (10U/ul) | ||
| align="center" | 0.2 | | align="center" | 0.2 | ||
| align="center" | 3. | | align="center" | 3.2 | ||
|- style="font-size:8pt" align="center | |- style="font-size:8pt" align="center" | ||
|style="font-weight:bold" height=" | |style="font-weight:bold" height="15" | Ampligase (5U/ul) | ||
| align="center" | 0.2 | | align="center" | 0.2 | ||
| align="center" | 3. | | align="center" | 3.2 | ||
|- style="font-size:8pt" align="center | |- style="font-size:8pt" align="center" | ||
|style="font-weight:bold" height=" | |style="font-weight:bold" height="15" | H2O | ||
| align="center" | 0.1 | | align="center" | 0.1 | ||
| align="center" | 1. | | align="center" | 1.6 | ||
|- style="font-size:8pt" align="center | |- style="font-size:8pt" align="center" | ||
| align="center" height=" | | align="center" height="15" | | ||
| align="center" | | |||
| align="center" | | | align="center" | | ||
|- style=" | |- style="font-size:8pt;font-weight:bold" align="center" | ||
|style=" | |style="text-decoration:underline" height="15" | SLN Mix for 50ul Rxn | ||
|style=" | |style="text-decoration:none" | 1x | ||
| | | 11x | ||
|- style="font-size:8pt" align="center | |- style="font-size:8pt" align="center" | ||
|style="font-weight:bold" height=" | |style="font-weight:bold" height="15" | dNTP (10uM) | ||
| align="center" | 1.25 | | align="center" | 1.25 | ||
| align="center" | | | align="center" | 13.75 | ||
|- style="font-size:8pt" align="center | |- style="font-size:8pt" align="center" | ||
|style="font-weight:bold" height=" | |style="font-weight:bold" height="15" | Stoffel (10U/ul) | ||
| align="center" | 0.5 | | align="center" | 0.5 | ||
| align="center" | | | align="center" | 5.5 | ||
|- style="font-size:8pt" align="center | |- style="font-size:8pt" align="center" | ||
|style="font-weight:bold" height=" | |style="font-weight:bold" height="15" | Ampligase (5U/ul) | ||
| align="center" | 0.5 | | align="center" | 0.5 | ||
| align="center" | | | align="center" | 5.5 | ||
|- style="font-size:8pt" align="center | |- style="font-size:8pt" align="center" | ||
|style="font-weight:bold" height=" | |style="font-weight:bold" height="15" | H2O | ||
| align="center" | 0.25 | | align="center" | 0.25 | ||
| align="center" | | | 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:# | |- style="background-color:#FFFF00;font-size:8pt;font-weight:bold" align="center" | ||
|style=" | |style="text-decoration:underline" width="81" height="15" | Exo I & III for 20ul Rxn | ||
|style=" | |style="text-decoration:none" width="112" | 1X | ||
| width=" | | width="83" | 16x | ||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
| height=" | | height="15" | Exo I (20U/ul) | ||
| align="center" | 1 | | align="center" | 1 | ||
| align="center" | 16 | | align="center" | 16 | ||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
| height=" | | height="15" | ExoIII (200U/ul) | ||
| align="center" | 1 | | align="center" | 1 | ||
| align="center" | 16 | | align="center" | 16 | ||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
| align="center" height=" | | align="center" height="15" | | ||
| align="center" | | | align="center" | | ||
| align="center" | | | align="center" | | ||
|- style="background-color:# | |- style="background-color:#FFFF00;font-size:8pt;font-weight:bold" align="center" | ||
|style=" | |style="text-decoration:underline" height="15" | Exo I & III for 50ul Rxn | ||
|style=" | |style="text-decoration:none" | 1X | ||
| | | 11x | ||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
| height=" | | height="15" | Exo I (20U/ul) | ||
| align="center" | 2.5 | | align="center" | 2.5 | ||
| align="center" | | | align="center" | 6.25 | ||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
| height=" | | height="15" | ExoIII (200U/ul) | ||
| align="center" | 2.5 | | align="center" | 2.5 | ||
| align="center" | | | 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" | | |||
| align="center" width="65" | | |||
|- 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" | | |||
| align="center" | | |||
|- 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" | | |||
| align="center" | | |||
|- 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" | | |||
| align="center" | | |||
|- 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" | | |||
| align="center" | | |||
|- 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" | | |||
| align="center" | | |||
|- style="font-size:8pt" align="center" | |||
| height="11" | SYBR Green 50X | |||
| align="center" | 0.4 | |||
| align="center" | 4.4 | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
|- style="font-size:8pt" align="center" | |||
| align="center" height="11" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
|- 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" | | |||
| align="center" | | |||
|- 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" | | |||
| align="center" | | |||
|- 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" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
|- style="font-size:8pt" align="center" | |||
| align="center" height="11" | | |||
| align="center" | | |||
| align="center" | | |||
| H16 | |||
| align="center" | 21 | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
|} | |} | ||
95C 2min -> (98C 20S -> 58C 20S -> 72C 20S) x 6 -> (98C 10S -> 72C 20S) x40 | |||
95C 2min -> | Monitor the reactions and terminate the program right before the amplification curves reach the plateau. | ||
==Indexing PCR for 3 samples== | |||
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.