Dinh/Dinh 2015/NOTES/2015-9-15
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1 ng RRBS protocol from plasma DNA[edit]
- Combined plasma DNA yielded 30 uL x 1.64 ng/uL
- Test different input: 2 ng, 1 ng, 0.5 ng, NTC. Perform duplicates of each.
- Mostly following Noi's protocol (Noi’s 1 ng input protocol from LabWiki: 2014-6-29).
MspI digestion[edit]
MspI (10U/uL) 1.00 uL 10x Tango Buffer 2.00 uL 1.2pg/ul unmeth lambda DNA 0.83 uL (should be 12pg/uL but I used the wrong one) 1 ng DNA input 6.00 uL (varying 0.5ng-2ng) Nuclease free water 8.17 uL
- Spin down the tubes, mix by gentle pulse vortexing on PCR 10x
- Spin down the tubes,
- Incubate 37C O/N (~13 hr) → 65C 20 min → hold at 4C
End-repair/dA-tailing[edit]
MspI digested DNA 18.00 uL Klenow exo- 1.00 uL dATP:dCTP:dGTP (20mM,2mM,2mM) 1.00 uL
- Spin down the tubes, mix by gentle pulse vortexing on PCR rack 10x
- Spin down the tubes,
- Incubate 30C 20 min → 37C 20 min → hold at 4C (no heated lid)
- Heat inactive 75C 10 min → 4C (with heated lid)
Methylated adaptor ligation[edit]
dA-tailed DNA 20.00 uL 10X Tango Buffer 0.50 uL HC T4 DNA ligase(30U/uL) 1.00 uL 10mM ATP 1.25 uL Nuclease free water 1.25 uL 1:20 diluted TruSeq adaptors 1.00 uL
- TruSeq barcodes
NTC_1 18 NTC_2 19 2ng_1 20 2ng_2 21 1ng_1 22 1ng_2 23 0.5ng_1 25 0.5ng_2 27
- Spin down the tubes, mix by gently pulse vortexing on PCR rack 10x
- Spin down the tubes,
- Incubate 16C, 22 h (no heated lid, >=20 hr)
- Mix by gentle pulse vortexing on PCR rack 5x
- Spin down
- Heat inactivate at 65C, 20 min → hold 4C
AMPure beads purification[edit]
- Need to confirm with Noi whether she used beads to purify the adaptor ligated DNA.
- Add 5 uL of water to make total volume equals 30 uL of ligation mixture.
- Add 60 uL of AMPure beads and repipette 10X to mix.
- Incubate for 30 minutes at room temperature.
- Wash twice with freshly prepared 70% ethanol.
- Dry for 15 minutes at room temperature. (While drying, label and UV new tubes for bisulfite conversion below).
- Add 30 uL of EB buffer to elute.
Bisulfite conversion using LifeTech MethylCode Bisulfite conversion kit[edit]
- Prepare 3 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer.
- If CT Conversion reagent prepare with 800ul H2O, need to add 5ul of H2O to 25ul adaptor-ligated DNA and added 120ul of CT conversion reagent.
- Mix well and spin down the tube
- Put on thermomixer at 37C for 15min, 1,000rpm
- Solution should be clear
- Spin down the tube
- Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA --> total volume 150ul
- Mix by pipetting 10X with multi-channel pipette
- Spin down the tube
- Incubate following below program
98°C for 10 minutes (DNA denaturation) 64°C for 2.5 hours (Bisulfite conversion) 4°C storage for up to 20 hours or continue to desulfonation
- Add 600ul Binding Buffer to each column
- Adjust p200 to 200ul and transfer bisulfite treated DNA to the column
- Mix by pipetting 5x
- Spin down column at 14,000 rpm for 30s
- Discard spnt
- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube)
- Spin down column at 14,000 rpm for 30s (do not discard spnt)
- Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube). Incubate for 15-20 minutes at room temperature.
- Spin down column at 14,000 rpm for 30s (do not discard spnt)
- Add 200ul Wash Buffer
- Spin down column at 14,000 rpm for 30s
- Discard spnt
- Add 200ul Wash Buffer
- Spin down column at 14,000 rpm for 3min (do not discard spnt)
- Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transferring to new tube
- Add 38ul of Elution Buffer (warm Elution Buffer at 60C for 10min)
- Wait for 2min
- Spin down column at 12,000 rpm for 1min
- Continue to amplification (During elution step, PCR mix should be prepared)
Amplification[edit]
- Perform amplification with and without Sybr. I reduced reaction volumes by half each.
- Perform PCR with 12 cycles (fixed)
bis-cvt DNA 15.000 uL 10X Reaction buffer 2.500 uL dNTP, 10mM 0.625 uL TruSeq F/R, 10uM 0.750 uL 50X Sybr 0.400 uL (0 for no sybr) PfuTurboCX 0.500 uL Nuclease free water 5.225 uL (5.625 for no sybr)
- Mix well
- Program: 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 cycles --> 72C for 2min
- Purify with 1X AMPure beads, incubate DNA with beads 15 minutes, wash twice with freshly prepared 80% ethanol, dry beads for 20 minutes (?))
- Elute with 20 uL EB buffer
- Continue to 2nd round PCR. No PAGE Verification
- Test 2nd round PCR
- Noi's protocol:
Purified 1st round DNA 3.00 uL 5X Phusion HF buffer 2.00 uL dNTP mix, 10mM 0.25 uL TruSeq F/R, 10 uM 0.30 uL 50X Sybr 0.08 uL (0 for no sybr) Phusion HF 0.10 uL Nuclease free water 4.27 uL (4.35 for no sybr)
- I couldn't find Phusion HF, so I used Phusion Hotstart Flex Master Mix instead.
- Mix well
- Program: 98C for 2min → [98C for 10sec → 60C for 30sec → 72C for 1min] X 9X cycles → 72C for 2min
- Load everything to 6% gel and run at 225 V for 30 min.
- Once PCR cycle is determined (8-9 cycles?), prepare PCR for all the samples and then purify with AMPure beads. Run gel to also determine the pooling ratios and perform size selection for 150-400 bp bands.
- Gel images:
- (+) is with sybr added, (-) is without sybr added. The amplification patterns are similar on gel, except in some cases sybr added more intensity.
- I remember that PfuTurboCx have less amplification with sybr, its not possible that sybr would increase the amplification efficiency, so I think the sybr green may have helped to make the fluorescence brighter for the DNA amplified with sybr. For now, I don't see evidencee that the sybr have an effect on amplification, so I could amplify with or without sybr.
File:Dinh 1ng RRBS 09182015 gel1.jpg File:Dinh 1ng RRBS 09182015 gel2.jpg