>Alan6017518 |
>Alan6017518 |
(One intermediate revision by the same user not shown) |
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| Monitor the reactions and terminate the program right before the amplification curves reach the plateau. | | Monitor the reactions and terminate the program right before the amplification curves reach the plateau. |
| ==Indexing PCR for 3 samples== | | ==Indexing PCR for 3 samples== |
| ==Indexing PCR with Kapa SYBR Fast==
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| *Repeat PCR with same condition as first round of captured library in order to be consistance
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| *Qiaquick column purify the 3 libraries that were taken out at the early stage of 1st round amplification
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| *Elute with 30ul EB, use 15ul for amplification
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| {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
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| |- style="font-size:8pt" align="center"
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| |style="background-color:#FFFF00;font-weight:bold;text-decoration:underline" width="165" height="15" | Content (50ul Capture) 9 rxn
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| |style="background-color:#FFFF00;font-weight:bold" width="35" | Volume
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| |style="background-color:#FFFF00;font-weight:bold" width="70" | Master Mix
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| |style="background-color:#FFFF00;font-weight:bold" width="56" | ChIPK36me3
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| |style="background-color:#FFFF00;font-weight:bold" width="36" | Index
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| |style="background-color:#FFFF00;font-weight:bold" width="65" | ChIPK27me3
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| |style="background-color:#FFFF00;font-weight:bold" width="28" | Index
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| |style="text-decoration:none" align="center" width="65" |
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| | align="center" width="65" |
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|
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| |- style="font-size:8pt" align="center"
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| | height="15" | Captured DNA
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| | align="center" | 27
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| | Do not add to MM
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| | GM12878
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| | align="center" | 1
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| | GM12878
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| | align="center" | 10
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| | align="center" |
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| | align="center" |
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|
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| |- style="font-size:8pt" align="center"
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| | height="15" | 2X Kapa SYBG qPCR master mix
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| | align="center" | 50
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| | align="center" | 495
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| |style="color:#FF0000" | H0
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| | align="center" | 2
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| | H0
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| | align="center" | 11
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| | align="center" |
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| | align="center" |
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|
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| |- style="font-size:8pt" align="center"
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| | height="15" | 10uM AmpF6.3Sol
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| | align="center" | 4
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| | align="center" | 39.6
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| | H7
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| | align="center" | 3
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| | H7
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| | align="center" | 12
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| | align="center" |
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| | align="center" |
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|
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| |- style="font-size:8pt" align="center"
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| | height="15" | 10uM AmpR6.3Ind
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| | align="center" | 4
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| | Do not add to MM
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| | H14
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| | align="center" | 4
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| | H14
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| | align="center" | 13
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| | align="center" |
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| | align="center" |
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|
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| |- style="font-size:8pt" align="center"
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| | height="15" | H2O
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| | align="center" | 15
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| | align="center" | 148.5
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| | H16
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| | align="center" | 9
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| | H16
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| | align="center" | 14
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| | align="center" |
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| | align="center" |
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|
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| |- style="font-size:8pt" align="center"
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| | align="center" height="15" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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|
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| |- style="font-size:8pt" align="center"
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| | align="center" height="15" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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|
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| |- style="background-color:#3366FF;font-size:8pt;font-weight:bold" align="center"
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| |style="text-decoration:underline" height="15" | Content (20ul Capture) 13rxn
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| | Volume
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| | Master Mix
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| | WCE
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| | Index
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| | cDNA
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| | Index
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| | gDNA
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| | Index
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|
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| |- style="font-size:8pt" align="center"
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| |style="text-decoration:none" height="15" | Captured DNA
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| | align="center" | 11
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| | Do not add to MM
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| | GM12878
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| | align="center" | 15
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| |style="color:#FF0000" | GM
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| | align="center" | 22
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| | H7
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| | align="center" | 27
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|
| |
| |- style="font-size:8pt" align="center"
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| | height="15" | 2X Kapa SYBG qPCR master mix
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| | align="center" | 50
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| | align="center" | 715
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| | P10
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| | align="center" | 16
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| | H0
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| | align="center" | 23
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| | H14
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| | align="center" | 28
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|
| |
| |- style="font-size:8pt" align="center"
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| | height="15" | 10uM AmpF6.3Sol
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| | align="center" | 4
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| | align="center" | 57.2
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| |style="color:#FF0000" | DF2
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| | align="center" | 17
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| | H7
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| | align="center" | 24
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| | H16
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| | align="center" | 29
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|
| |
| |- style="font-size:8pt" align="center"
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| | height="15" | 10uM AmpR6.3Ind
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| | align="center" | 4
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| | Do not add to MM
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| | H0
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| | align="center" | 18
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| | H14
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| | align="center" | 25
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| | align="center" |
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| | align="center" |
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|
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| |- style="font-size:8pt" align="center"
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| | height="15" | H2O
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| | align="center" | 31
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| | align="center" | 443.3
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| | H7
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| | align="center" | 19
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| | H16
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| | align="center" | 26
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| | align="center" |
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| | align="center" |
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|
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| |- style="font-size:8pt" align="center"
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| | align="center" height="15" |
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| | align="center" |
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| | align="center" |
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| | H14
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| | align="center" | 20
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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|
| |
| |- style="font-size:8pt" align="center"
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| | align="center" height="15" |
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| | align="center" |
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| | align="center" |
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| | H16
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| | align="center" | 21
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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|
| |
| |- style="font-size:12pt" valign="bottom"
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| | align="center" height="15" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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| | align="center" |
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|
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| |- style="font-size:8pt" align="center"
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| |style="background-color:#CCFFCC;font-weight:bold;text-decoration:underline" height="15" | Content (15ul Capture) 3rxn
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| |style="background-color:#CCFFCC;font-weight:bold" | Volume
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| |style="background-color:#CCFFCC;font-weight:bold" | Master Mix
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| |style="background-color:#CCFFCC" | Type
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| |style="background-color:#CCFFCC;font-weight:bold" | Sample
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| |style="background-color:#CCFFCC;font-weight:bold" | Index
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| |style="font-weight:bold" align="center" |
| |
| |style="font-weight:bold" align="center" |
| |
| |style="font-weight:bold" align="center" |
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|
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| |-
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| |style="font-size:8pt;text-decoration:none" height="15" align="center" | Captured DNA
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| |style="font-size:8pt" align="center" align="center" | 15
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| |style="font-size:8pt" align="center" | Do not add to MM
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| |style="background-color:#FFFF00;font-size:8pt;font-weight:bold;text-decoration:underline" align="center" | ChIPK36me3
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| |style="font-size:8pt;text-decoration:none;color:#FF0000" align="center" | H0
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| |style="font-size:8pt" align="center" align="center" | 2
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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|
| |
| |-
| |
| |style="font-size:8pt" height="15" align="center" | 2X Kapa SYBG qPCR master mix
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| |style="font-size:8pt" align="center" align="center" | 50
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| |style="font-size:8pt" align="center" align="center" | 165
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| |style="background-color:#3366FF;font-size:8pt;font-weight:bold;text-decoration:underline" align="center" | WCE
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| |style="font-size:8pt;text-decoration:none;color:#FF0000" align="center" | DF2
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| |style="font-size:8pt" align="center" align="center" | 17
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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|
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| |-
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| |style="font-size:8pt" height="15" align="center" | 10uM AmpF6.3Sol
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| |style="font-size:8pt" align="center" align="center" | 4
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| |style="font-size:8pt" align="center" align="center" | 13.2
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| |style="background-color:#3366FF;font-size:8pt;font-weight:bold;text-decoration:underline" align="center" | cDNA
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| |style="font-size:8pt;text-decoration:none;color:#FF0000" align="center" | GM
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| |style="font-size:8pt" align="center" align="center" | 22
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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|
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| |-
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| |style="font-size:8pt" height="15" align="center" | 10uM AmpR6.3Ind
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| |style="font-size:8pt" align="center" align="center" | 4
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| |style="font-size:8pt" align="center" | Do not add to MM
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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|
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| |-
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| |style="font-size:8pt" height="15" align="center" | H2O
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| |style="font-size:8pt" align="center" align="center" | 27
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| |style="font-size:8pt" align="center" align="center" | 89.1
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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| |style="font-size:12pt" align="center" valign="bottom" |
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|
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| |}
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| *The 3 samples highlighted in red will use 15ul from the 1st round PCR (purified product) as the 2nd round PCR is already amplified with Kapa HiFi.
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| *First add Index primer
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| *Then add captured DNA
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| *Prepare Master Mix
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| 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 6 -> (98C 10S -> 72C 20S) x40
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| Monitor the reactions and terminate the program right before the amplification curves reach the plateau.
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| *Master mix for 50ul reaction was frozen on the ice block, I realized this after adding in the MM to wells, ran pcr nothing amplified based on the PCR curve.
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| *Qiaquick elute with 29ul
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| *Redo amplification
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| {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
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| |- style="background-color:#FFFF00;font-size:8pt;font-weight:bold" align="center"
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| |style="text-decoration:underline" width="165" height="15" | Content (50ul Capture) 10 rxn
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| | width="35" | Volume
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| | width="70" | Master Mix
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| | width="56" | ChIPK36me3
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| | width="36" | Index
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| | width="65" | ChIPK27me3
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| | width="28" | Index
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|
| |
| |- style="font-size:8pt" align="center"
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| |style="text-decoration:none" height="15" | Captured DNA
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| | align="center" | 27
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| | Do not add to MM
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| | GM12878
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| | align="center" | 1
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| | GM12878
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| | align="center" | 10
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|
| |
| |- style="font-size:8pt" align="center"
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| | height="15" | 2X Kapa SYBG qPCR master mix
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| | align="center" | 50
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| | align="center" | 550
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| |style="color:#FF0000" | H0
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| | align="center" | 2
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| | H0
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| | align="center" | 11
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|
| |
| |- style="font-size:8pt" align="center"
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| | height="15" | 10uM AmpF6.3Sol
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| | align="center" | 4
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| | align="center" | 44
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| | H7
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| | align="center" | 3
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| | H7
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| | align="center" | 12
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|
| |
| |- style="font-size:8pt" align="center"
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| | height="15" | 10uM AmpR6.3Ind
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| | align="center" | 4
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| | Do not add to MM
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| | H14
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| | align="center" | 4
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| | H14
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| | align="center" | 13
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|
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| |- style="font-size:8pt" align="center"
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| | height="15" | H2O
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| | align="center" | 15
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| | align="center" | 165
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| | H16
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| | align="center" | 9
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| | H16
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| | align="center" | 14
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|
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| |}
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|
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| ==Purification & Quantification==
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| *Take 1ul to runa TBE PAGE gel
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| [[File:ZhangLab_2 2012-08-18 05hr 02min.jpg|400px]]
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| [[File:ZhangLab_2 2012-08-18 05hr 00min.jpg|400px]]
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| *Qiaquick purify all libraries, elute with 30ul EB
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| *Qubit quantification
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| ==Pool library 1-10==
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| ==Pool Library 11-25==
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