AlanFung:LabNotes/CTCF/2011-7-28: Difference between revisions

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>Alan6017518
 
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=Low-input Bisulfite Conversion=
=Low-input Bisulfite Conversion=
* I will be working on the Tagmentation first protocol.
* I will be working on the bisulfite conversion first protocol.
==Protocols Today==
==Protocols Today==
* Start with four tubes: 1571 cells, 314 cells, 157 cells, and NTC
* Start with four tubes: 1571 cells, 314 cells, 157 cells, NTC, and cells that do not go through bisulfite conversion
===Cell Lysis===
===Cell Lysis===
* First, make protease Dilution
* First, make protease Dilution
**Add 3 uL Protease to 97 uL H2O for a 3:100 dilution
**Add 3 uL Protease to 97 uL H2O for a 3:100 dilution
**Add 1 uL Protease to 99 uL H2O for a 1:100 dilution
**Add 1 uL Protease to 99 uL H2O for a 1:100 dilution
* Next, add 1 uL 3:100 diluted Protease and 1 uL Buffer to 1 uL Frozen cells; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
* 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
* Cells are now lysed
===Bisulfite Conversion - Imprint Kit===
===Bisulfite Conversion - Imprint Kit===
* Prepare DNA Modification Solution (CT Conversion Reagent):  
* Prepare DNA Modification Solution (CT Conversion Reagent):  
* Add 16 uL 0.5 mg/mL BSA and 1 uL Imprint Balance solution to each sample; incubate at 37 C for 10 minutes
* Add 21 uL 0.5 mg/mL BSA and 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
* Add 125 uL DNA Modification solution to each tube, incubate at 65 C for 90 minutes
* Perform Column Purification of each sample:
* Perform Column Purification of each sample:
** XXX
** Elute into 10 uL Elution Buffer
** Elute into 10 uL Elution Buffer
===MDA===
 
===MDA - Epitect Kit===
*Denature the no bisulfite conversion control by heating at 95C for 30 sec and cool down immediately with a cold block
*Thaw REPLI-g Midi DNA Polymerase on ice. Thaw all other components at room temperature, vortex, then centrifuge briefly.
*Thaw REPLI-g Midi DNA Polymerase on ice. Thaw all other components at room temperature, vortex, then centrifuge briefly.
*Add 1ul REPLI-g Midi DNA Polymerase to 29ul WBA reaction buffer
*Add 1ul REPLI-g Midi DNA Polymerase to 29ul WBA reaction buffer
Line 22: Line 24:
*Add 30ul Master mix to 10ul of bisulfite converted DNA
*Add 30ul Master mix to 10ul of bisulfite converted DNA
*Incubate at 28C for 1 h and 95C for 5 min
*Incubate at 28C for 1 h and 95C for 5 min
==Ethanol Precipitation ==
*Add 2.5X 100% Ethanol
*Add 0.1X 3M NaOAC
*Add 1ul glycoblue
*Store at -80C for 30min
*Spin down at 10,000rpm at 4C for 15min
*Remove all liquid
*Add 500ul 4C chilled 75% ethanol
*Spin at 10,000rpm at 4C for 5 min
*Remove all liquid
*Let dry in hood for 5 min
*Add 10ul of nuclease free H2O (aliquot 2ul to perform PCR Analysis)
*Let sit at RT to dissolve the pellet


===Tagmentation===
===MinElute ===
*Elute with 12ul buffer
===Nanodrop===
*Perform nanodrop  on a few of the samples
*1500cells-12.9ng/ul
*150cells(NBS)-31.8ng/ul
*1500cells(NBS)-48ng/ul
 
===Tagmentation -  Shendure Protocol===
*Take 3ul of the MDA product for 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 lysate, add 1 uL 1:50 diluted Nextera Transposase and 1 uL 5x Nextera HMW Buffer; incubate at 55 C for 5 minutes
* Next, add 1 uL 1:100 diluted Protease to solution; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
* Next, add 1 uL 1:100 diluted Protease to solution; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
* To this volume, add 1 uL 10 U/uL Klenow exo- Polymerase and 1 uL of methylated dNTPs (1 mM); incubate at 30 C for 30 minutes
*Store at -20C
* Library is now ready for Bisulfite Conversion


===Amplification===
===Amplification - Kapa + BST===
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Content'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''MM'''
|-
| Nuclease Free H2O||16||140.8
|-
| 10uM Orange Primer||1||8.8
|-
| 10uM Blue Primer||1||8.8
|-
| Tagmented DNA||6||Do not add to MM
|-
| 2X Kapa sybr supermix||25||220
|-
| BST||1||8.8
|-
| Total||50||387.2
|}
*Transfer DNA to strip tube and add 44ul MM to each reaction
* Raise volume of any non-bisulfite converted samples to 10 uL (add 2 uL H2O)
* Raise volume of any non-bisulfite converted samples to 10 uL (add 2 uL H2O)
* Add 1 uL Orange Primer, 1 uL Blue Primer, 25 uL KAPA SYBR Supermix, 1 uL BST Polymerase or Klenow Polymerase, and 12 uL H2O to each sample
* Add 1 uL Orange Primer, 1 uL Blue Primer, 25 uL KAPA SYBR Supermix, 1 uL BST Polymerase or Klenow Polymerase, and 12 uL H2O to each sample
Line 55: Line 72:
** 72 C, 5 minutes (final extension)
** 72 C, 5 minutes (final extension)
** Hold at 4 C
** Hold at 4 C
===Sequencing===
 
* Hopefully, libraries will pop up as expected.
===PAGE Gel Analysis===
* Should probably try some "no-bisulfite" controls...
[[File:ZhangLab_2 2011-07-29 14hr 42min.jpg|500px]]
* Would need to realiquot cells today; six additional aliquots
 
* Talk to Alan.
===Results===
[[File:072911.jpg]]
*Shouldn't have heat inactivates the MDA product as that will denature the DNA and makes it single stranded.
*I can probably skip the inactivation step and just perform a column purification to eliminate the polymerase

Latest revision as of 22:59, 29 July 2011

Low-input Bisulfite Conversion[edit]

  • I will be working on the bisulfite conversion first protocol.

Protocols Today[edit]

  • Start with four tubes: 1571 cells, 314 cells, 157 cells, NTC, and cells that do not go through bisulfite conversion

Cell Lysis[edit]

  • First, make protease Dilution
    • Add 3 uL Protease to 97 uL H2O for a 3:100 dilution
    • Add 1 uL Protease to 99 uL H2O for a 1:100 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

Bisulfite Conversion - Imprint Kit[edit]

  • Prepare DNA Modification Solution (CT Conversion Reagent):
  • Add 21 uL 0.5 mg/mL BSA and 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 10 uL Elution Buffer

MDA - Epitect Kit[edit]

  • Denature the no bisulfite conversion control by heating at 95C for 30 sec and cool down immediately with a cold block
  • Thaw REPLI-g Midi DNA Polymerase on ice. Thaw all other components at room temperature, vortex, then centrifuge briefly.
  • Add 1ul REPLI-g Midi DNA Polymerase to 29ul WBA reaction buffer
  • Mix and centrifuge briefly
  • Add 30ul Master mix to 10ul of bisulfite converted DNA
  • Incubate at 28C for 1 h and 95C for 5 min

MinElute[edit]

  • Elute with 12ul buffer

Nanodrop[edit]

  • Perform nanodrop on a few of the samples
  • 1500cells-12.9ng/ul
  • 150cells(NBS)-31.8ng/ul
  • 1500cells(NBS)-48ng/ul

Tagmentation - Shendure Protocol[edit]

  • Take 3ul of the MDA product for 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
  • Next, add 1 uL 1:100 diluted Protease to solution; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
  • Store at -20C

Amplification - Kapa + BST[edit]

Content Volume MM
Nuclease Free H2O 16 140.8
10uM Orange Primer 1 8.8
10uM Blue Primer 1 8.8
Tagmented DNA 6 Do not add to MM
2X Kapa sybr supermix 25 220
BST 1 8.8
Total 50 387.2
  • Transfer DNA to strip tube and add 44ul MM to each reaction
  • Raise volume of any non-bisulfite converted samples to 10 uL (add 2 uL H2O)
  • Add 1 uL Orange Primer, 1 uL Blue Primer, 25 uL KAPA SYBR Supermix, 1 uL BST Polymerase or Klenow Polymerase, and 12 uL H2O to each sample
  • Perform qPCR using the following reaction conditions:
    • 60 C, 20 min incubation (BST second strand synthesis)
    • 72 C, 3 min incubation (Nextera extension (where necessary; not needed if using Klenow))
    • 95 C, 30 seconds (Denature)
    • 35 cycles of:
      • 95 C, 10 seconds (Denature)
      • 58 C, 30 seconds (Anneal)
      • 72 C, 3 minutes (Extend)
    • 72 C, 5 minutes (final extension)
    • Hold at 4 C

PAGE Gel Analysis[edit]

File:ZhangLab 2 2011-07-29 14hr 42min.jpg

Results[edit]

File:072911.jpg

  • Shouldn't have heat inactivates the MDA product as that will denature the DNA and makes it single stranded.
  • I can probably skip the inactivation step and just perform a column purification to eliminate the polymerase