AlanFung:LabNotes/CTCF/2011-10-4
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Notes[edit]
- From previous experiment, it is verified that cells can be digested using NaOH. I will repeat experiment with the following improvements.
- NaOH cell digestion, avoid using NP40 and protease, save time and effort.
- EtOH precipitation after MDA, prevent size elimination with column purification and avoid spending time to speedvac to cut down the sample volume for tagmentation
Protocols Today[edit]
- 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 & Bisulfite Conversion[edit]
150cells & Negative control[edit]
- Add 23ul 0.5 mg/mL BSA
- Add 1ul imprint Balance solution to each sample (150 GM12878cells/ul & ddH2O)
- 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
MDA[edit]
- Denature all samples at 95C for 30sec and quickly transfer to cooler rack
Content | Volume | MM |
10X Repli Phi Buffer | 2 | 6.2 |
1mM N9 Buffer | 1 | 3.1 |
25mM dNTP | 0.8 | 2.48 |
Repliphi Phi 29 (100U/ul) | 1 | 3.1 |
Klenow Exo Minus (10U/ul) | 0.5 | 1.55 |
Nuclease free H2O | 4.7 | 14.57 |
Modified DNA Sample | 10 | Do not add to MM |
Total | 20 | 31 |
- Add 10ul of MDA mastermix to each sample
- 30C 60 min, 65C 10 min, 4C hold
EtOH Precipitation[edit]
- Elute with 3ul nuclease free water
- Incubate at RT for at least 10min
- Transfer samples to qPCR strip tubes for tagmentation and qPCR
Tagmentation[edit]
- 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)
- 20 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
- Stopped reaction at cycle 17
- Purify with minelute, elute with 15ul of EB buffer, keep 1ul for gel
Bisulfite Verification w CHR8,21&22[edit]
MM | CHR8 | CHR21 | CHR22 |
2X iQ Super Mix | 20 | 20 | 20 |
Primer F (3.3uM) | 6 | 6 | 6 |
Primer R (3.3uM) | 6 | 6 | 6 |
gDNA | 2 | 2 | 2 |
H2O | 6 | 6 | 6 |
Total | 40 | 40 | 40 |
Step1 96C, 3m Step2 95C, 30s Step3 62C, 1m Step4 72C, 1m Step5 Go to step2 repeat 39 times Step6 72C, 5m Step7 4C, Forever
Adding Purple and Pink Nextera Adapters[edit]
- Using barcode 8 here
Content | Volume Per Reaction |
DNA | Do not add to MM |
Kapa HF Mix | 55 |
Adaptor 1 | 2.2 |
Adapor 2 (Barcode) | 2.2 |
H2O | 17.6 |
Total | 77 |
- Added 0.4ul 50X SYBR Green to each reaction
Nextera_2nd 95C 30sec [95C 10sec; 60C 30sec; 72C 2min] X 2 [95C 10sec; 62C 30sec; 72C 2min] X 3 72C, 3min
Page Gel Analysis[edit]
- I will run a gel with the CHR8,21,22 samples and the blue/orange primers amplified samples w. -ve control
Results[edit]
- The bisulfite conversion pcr test failed for some reason. Mostly due to old sodium bisulfite reagent. It was stored at -20C as recommended, but it is better to work with freshly prepared bisulfite conversion reagents. Will spend more time to investigate on this if possible.
- The 1st amplification worked out really nice, with the exact amplicon size as expected and negative control didn't get amplified.
- However after the 2nd amplification, unspecific bands showed up for both the sample and the control. I suspect this is due to lost of samples due to purification using the column.
- I would like to try Rui's protocol where she did purification with beads and she had pretty good results from it.