AlanFung:LabNotes/2014/2014-9-8: Difference between revisions

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>Alan6017518
(Created page with "===qPCR Test to verifiy End repair, A-tailing & Adapter Ligation=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconvers...")
 
>Alan6017518
 
(8 intermediate revisions by the same user not shown)
Line 14: Line 14:
| height="14" | Adapter ligated DNA
| height="14" | Adapter ligated DNA
| align="center" | 1
| align="center" | 1
| align="center" | 19.57
| align="center" | 19.6


|-  align="center"
|-  align="center"
| height="14" | 10uM PCR_F
| height="14" | 10uM PCR_F
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 9.785
| align="center" | 9.8


|-  align="center"
|-  align="center"
| height="14" | 10uM PCR_R.Ind2
| height="14" | 10uM PCR_R.Ind2
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 9.785
| align="center" | 9.8


|-  align="center"
|-  align="center"
| height="14" | 2x iQ MM
| height="14" | 2X Kapa Fast
| align="center" | 12.5
| align="center" | 12.5
| align="center" | 244.625
| align="center" | 244.6


|-  align="center"
|-  align="center"
| height="14" | Water
| height="14" | Water
| align="center" | 10.5
| align="center" | 10.5
| align="center" | 205.485
| align="center" | 205.5


|-  align="center"
|-  align="center"
| height="14" | Total master mix volume
| height="14" | Total master mix volume
| align="center" | 25
| align="center" | 25
| align="center" | 489.25
| align="center" | 489.3


|}
|}
*98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles --> 72C 3min -->  15C Hold
[[File:09082014_Cycle12.JPG]]
==PAGE Gel Verification==
[[File:ZhangLab_2 2014-09-08 16hr 52min.jpg]]
*Bands ~110bp is most likely primer dimers, it was not removed by the beads as 1.7X beads will only remove anything below 100bp
*pick a few samples and Repeat reaction with 7,8,9 cycle
*Run PAGE gel with negative control and

Latest revision as of 23:24, 9 September 2014

qPCR Test to verifiy End repair, A-tailing & Adapter Ligation[edit]

Component 1 Library 19 Libraries + 3%
qPCR Master Mix:    
Adapter ligated DNA 1 19.6
10uM PCR_F 0.5 9.8
10uM PCR_R.Ind2 0.5 9.8
2X Kapa Fast 12.5 244.6
Water 10.5 205.5
Total master mix volume 25 489.3
  • 98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles --> 72C 3min --> 15C Hold

File:09082014 Cycle12.JPG

PAGE Gel Verification[edit]

File:ZhangLab 2 2014-09-08 16hr 52min.jpg

  • Bands ~110bp is most likely primer dimers, it was not removed by the beads as 1.7X beads will only remove anything below 100bp
  • pick a few samples and Repeat reaction with 7,8,9 cycle
  • Run PAGE gel with negative control and