AlanFung:LabNotes/CTCF/2011-7-28: Difference between revisions
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=Low-input Bisulfite Conversion= | =Low-input Bisulfite Conversion= | ||
* I will be working on the | * 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 | * 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 | * 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: | ||
** 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 | ||
===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 | ||
* | *Store at -20C | ||
===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 | ||
=== | |||
===PAGE Gel Analysis=== | |||
[[File:ZhangLab_2 2011-07-29 14hr 42min.jpg|500px]] | |||
* | |||
* | ===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]
- 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