AlanFung:LabNotes/CTCF/2011-8-26: Difference between revisions
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===Tagmentation=== | ===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 | ||
*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 | * 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 | *Prepare mastermix for library amplification |
Revision as of 05:37, 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
- 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 | 17 | 74.8 |
10uM Orange Primer | 1 | 4.4 |
10uM Blue Primer | 1 | 4.4 |
Tagmented DNA | 5 | Do not add to MM |
2X Kapa SYBR Supermix | 25 | 110 |
BST | 1 | 4.4 |
Total | 50 | 198 |
- Add 45ul MM to each reaction
- 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