Noi/NOTES/2014-7-22: Difference between revisions

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== 1st round amplification ==
* Perform 1st round PCR as previously described for 1ng low-input DNA
== 2nd round amplification (TEST) ==
* Did 2nd round PCR in small volume at 6 and 8 cycles and verified in 6% TBE gel as in previous experiment
== 2nd round amplification in a large volume ==
* I did 2nd PCR in larger volume (50ul X 2)
** 6 cycles for the samples with high intensity in TEST PCR and 7X for those samples with very low signal
* Purified 2nd PCR 90ul with 1X AMPure beads and eluted with 55ul EB buffer
* PAGE quantification in 6% TBE gel, volume 5ul
==== PAGE quantification results ====
* Gel1: Sample NC-P-12 - 21 (10 samples)
* Gel 2: Sample NC-P-22-30 (8 samples)
[[File:ZhangLab_2 2014-07-25 17hr 03min_PQ_dRRBS_NC-P_12-21.jpg| 500px]] [[File:ZhangLab_2 2014-07-25 17hr 03min_PQ_dRRBS_NC-P_22-30.jpg| 500px]]

Revision as of 19:58, 25 August 2014

Preparation of low-input RRBS sequencing libraries with MspI/TaqI double-digestion on normal plasma DNA

Calendar

  • Continued from [[1]]



  • I will add experimental details.

3) Methylated adaptor ligation

#strip/tube Code TruSeq Index
#1_1 NC-P-12 1
#1_2 NC-P-13 2
#1_3 NC-P-14 4
#1_4 NC-P-15 5
#1_5 NC-P-16 6
#1_6 NC-P-17 7
#1_7 NC-P-18 8
#1_8 NC-P-19 10
#2_1 NC-P-20 13
#2_2 NC-P-21 14
#2_3 NC-P-22 15
#2_4 NC-P-23 16
#2_5 NC-P-24 27
#2_6 NC-P-25 18
#2_7 NC-P-26 19
#2_8 NC-P-27 20
#3_1 NC-P-29 11
#3_2 NC-P-30 12
#3_3 NTC1 21
#3_4 NTC2 22

1st round amplification

  • Perform 1st round PCR as previously described for 1ng low-input DNA

2nd round amplification (TEST)

  • Did 2nd round PCR in small volume at 6 and 8 cycles and verified in 6% TBE gel as in previous experiment

2nd round amplification in a large volume

  • I did 2nd PCR in larger volume (50ul X 2)
    • 6 cycles for the samples with high intensity in TEST PCR and 7X for those samples with very low signal
  • Purified 2nd PCR 90ul with 1X AMPure beads and eluted with 55ul EB buffer
  • PAGE quantification in 6% TBE gel, volume 5ul

PAGE quantification results

  • Gel1: Sample NC-P-12 - 21 (10 samples)
  • Gel 2: Sample NC-P-22-30 (8 samples)

File:ZhangLab 2 2014-07-25 17hr 03min PQ dRRBS NC-P 12-21.jpg File:ZhangLab 2 2014-07-25 17hr 03min PQ dRRBS NC-P 22-30.jpg