AlanFung:LabNotes/CTCF/2011-8-26: Difference between revisions

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>Alan6017518
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** Hold at 4 C
** Hold at 4 C
*300 cells was taken out after 7 cycles
*300 cells was taken out after 7 cycles
*150 cells was taken out after 12 cycles
*150 cells & NTC was taken out after 12 cycles


===Purification===
===Purification===

Revision as of 22:21, 29 August 2011

Low-input Bisulfite Conversion and MDA Nextera library

Protocols Today

  • Start with four tubes: 314 cells, 157 cells x 2, NTC all go through bisulfite conversion
  • Make 1571 cell/ul (original tube)
  • Make 314 cells/ul (dilute 10ul cells in 40 ul ddh2o)
  • Make 157cells/ul (dilute 10ul cells in 90 ul ddh2o)

Cell Lysis

  • First, make protease Dilution
    • Add 3 uL Protease to 97 uL H2O for a 3:100 dilution
  • Next, add 1 uL 3:100 diluted Protease and 1 uL 3X Lysis Buffer (NP40) to 1 uL Frozen cells; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
  • Cells are now lysed

Make New BSA-Water solution

  • Stock solution is at 10mg/ml, need to dilute to 0.5mg/ml
  • Take 10ul stock BSA dilute with 190ul nuclease free water

Bisulfite Conversion - Imprint Kit

  • Prepare DNA Modification Solution (CT Conversion Reagent):
  • Add 21 uL 0.5 mg/mL BSA and
  • Add 1 uL Imprint Balance solution to each sample;
  • incubate at 37 C for 10 minutes
  • Add 125 uL DNA Modification solution to each tube, incubate at 65 C for 90 minutes
  • Perform Column Purification of each sample:
  • Elute into 11uL Elution Buffer

Continue on 8/28

  • Denature all samples at 95C for 30sec and quickly transfer to cooler rack
Content Volume MM
10X Repli Phi Buffer 2 8.8
1mM N9 Primer 1 4.4
25mM dNTP 0.8 3.52
Repliphi Phi 29 (100U/ul) 1 4.4
Klenow Exo Minus (10U/ul) 0.5 2.2
Nuclease free H20 4.7 20.68
Modified DNA Sample 10 Do not add to MM
Total 20 44
  • Add 10ul of MDA mastermix to each sample
  • 30C 60 min, 65C 10 min, 4C hold

MinElute

  • Elute with 10ul nuclease free water

Speedvac

  • To get 3ul for shendure's method of nextera library tagmentation, I will transfer the 10ul samples to qpcr tube, put the cap on and puncture a hole at the cap and speedvac till the sample is dry
  • took about 28min to dry samples
  • Re-dissolve the samples with 3ul of nuclease free water, sit at RT for 5 min

Tagmentation

  • To lysate, add 1 uL 1:50 diluted Nextera Transposase and 1 uL 5x Nextera HMW Buffer; incubate at 55 C for 5 minutes
  • To make 1:100 Protease, mix 1ul of protease with 99ul of nuclease free water
  • Next, add 1 uL 1:100 diluted Protease to solution; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
  • Prepare mastermix for library amplification
Content Volume MM
Nuclease Free H2O 16 70.4
10uM Orange Primer 1 4.4
10uM Blue Primer 1 4.4
Tagmented DNA 6 Do not add to MM
2X Kapa SYBR Supermix 25 110
BST 1 4.4
Total 50 193.6
  • Add 44ul MM to each reaction
  • Perform qPCR using the following reaction conditions:
    • 65 C, 3 min incubation (BST gap filling)
    • 95 C, 30 seconds (Denature)
    • 35 cycles of:
      • 95 C, 10 seconds (Denature)
      • 58 C, 30 seconds (Anneal)
      • 72 C, 3 minutes (Extension)
    • 72 C, 5 minutes (final extension)
    • Hold at 4 C
  • 300 cells was taken out after 7 cycles
  • 150 cells & NTC was taken out after 12 cycles

Purification

  • Minelute column, elute with 15ul water
  • Nanodrop gave me weird results, I was having problem when measuring the blank sterile water. Will avoid using the water provided by the Repliphi phi 29 kit for elution next time
    • 150:6.6ng/ul
    • 300:-0.2ng/ul
  • Use Qubit to measure the concentration instead
    • 150:4.16ng/ul
    • 300:1.46ng/ul
  • Looks like I do not have enough samples for end repair, may be I should run a few more cycles of PCR?
  • I would like to run a gel to confirm the size of the amplicons

Continue on 8/30