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