Noi/NOTES/2014-12-11: Difference between revisions

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=== TEST PCR ===
=== TEST PCR ===
:- Prepare PCR in total 20ul reaction with 4 ul BIS-DNA
:- Prepare PCR in total 20ul reaction with 4 ul BIS-DNA
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc'''
| align="center" style="width:60px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.'''
| align="center" style="width:60px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''10.5 rxn mix'''
|-
| Bis-cvt DNA||||||||||3.00||0.00
|-
| 2X KAPA HiFi Uracil HotStart ReadyMix||2||X||1||X||5.00||52.50
|-
| TruS_F/R||10||uM||0.3||uM||0.30||3.15
|-
| 50X SYBG||50||X||0.4||X||0.08||0.84
|-
| H2O||||||||||1.62||17.01
|-
| Total||||||||||10.00||
|}
:- Aliquot 3.5ul + 1.5ul BIS-DNA
::- ''98C for 45sec --> [98C for 15sec -> 60C for 30sec -> 72C for 30sec] X 13X --> 72C for 1min''
:- Verify all PCR product in 6% TBE gel
[[File:ZhangLab_2 2014-12-12 20hr 24min_RRBS-DZ-5ul.jpg|500px]]
* It looked like the pattern of size distribution was different from previous experiment as the size seemed to be more larger than sequencing library prepared from human tumor DNA.
* I continued to amplified the rest of BIS-DNA in 100ul reaction with cycle number 14 cycles for sample C1, C2, H1, H3, 15 cycles for samples C4, H4, H5 and 16 cycles for sample C3
:- Purified with 1X AMPure bead, elute with 32ul EB buffer
:- PAGE quantify by loading 4ul
[[File:ZhangLab_2 2014-12-15 19hr 52min_PQ_SeqLib-4ul.jpg| 500px]]

Revision as of 15:30, 16 December 2014

RRBS library prep, mouse Mouse hippocampus gDNA (Zhou's lab)

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  • 2014-12-01: Received 9 mouse hippocamplus gDNA from Zhou's lab

N.D. measurement by Orit

Sample ID ng/ul A260 A280 260/280 260/230
C1 113.72 2.274 1.199 1.9 2.32
C2 84.47 1.689 0.919 1.84 2.19
C3 109.47 2.189 1.207 1.81 1.79
C4 116.84 2.337 1.273 1.84 2.23
H1 115.85 2.317 1.285 1.8 2.14
H2 107.32 2.146 1.161 1.85 2.11
H3 106.12 2.122 1.14 1.86 2.11
H3 90.76 1.815 0.997 1.82 2.17
H4 145.22 2.904 1.68 1.73 1.29
H5 96.04 1.921 1.042 1.84 2.2
  • I re-quantified concentration by Qubit dsDNA HS assay using 1ul for quantification

Qubit dsDNA HS quantification result

Sample Conc. in the Qubit Unit uL used Dilution Conc. (ng/ul)
HC-C1 459 ng/mL 1 200 91.80
HC-C2 382 ng/mL 1 200 76.40
HC-C3 510 ng/mL 1 200 100.00
HC-C4 510 ng/mL 1 200 100.00
HC-H1 487 ng/mL 1 200 97.40
HC-H2 495 ng/mL 1 200 99.10
HC-H3 395 ng/mL 1 200 79.00
HC-H4 493 ng/mL 1 200 98.60
HC-H5 428 ng/mL 1 200 85.60

1) MspI digestion

  • I will use 500ng for RRBS library prep and spike in with 1ng of unmethylated lambda DNA for monitoring bisulfite conversion rate
  • Conditions: Digest 500ng of DNA with 20U of MspI in 30ul reaction at 37C 3h and heat inactivate at 65C 20min

MspI digestion set up

Sample ID Conc. (ng/ul) Volume for 500ng (ul) Sample code H2O MspI Mix Total (ul)
HC-C1 91.80 5.45 C1 4.55 20.00 30.00
HC-C2 76.40 6.54 C2 3.46 20.00 30.00
HC-C3 100.00 5.00 C3 5.00 20.00 30.00
HC-C4 100.00 5.00 C4 5.00 20.00 30.00
HC-H1 97.40 5.13 H1 4.87 20.00 30.00
HC-H2 99.10 5.05 H2 4.95 20.00 30.00
HC-H3 79.00 6.33 H3 3.67 20.00 30.00
HC-H4 98.60 5.07 H4 4.93 20.00 30.00
HC-H5 85.60 5.84 H5 4.16 20.00 30.00
NTC 0.00 0.00 NTC 10.00 20.00 30.00

Msp I reaction Mix

Components 1 rxn 10.5 rxn mix
MspI (10U/ul) 2.00 21.00
10X Tango Buffer 3.00 31.50
1ng/ul unmeth-lambda DNA 1.00 10.50
H2O 14.00 147.00
Total 20.00
- Add H2O to each tube
- Add 500ng of DNA to each tube
- Add 20ul of MspI reaction Mix to each tube and pipette few times
- Spin down the tubes
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tubes
- 37C, 3h --> 65C, 20min --> hold at 4C (set heat lid)


2) End-repair/dA-tailing

  • After MspI, I took 2ul of MspI-digested DNA for gel verification (equal to DNA ~33.33ng). Total volume left is 28ul
  • Mix 13ul of 5U/ul Klenow exo- (Thermoscientific) with 13ul of 20:2:2mM dAdCdG
  • Add 2ul of Klenow exo-/dAdCdG mix to each tube. Total volume is 30ul
- Spin down the tubes
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tubes
- 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- 75C, 10min --> hold at 4C (heat lid on)
- Perform AMPure bead purification with 2x volume AMPure beads

AMPure bead purification

- Add 60ul AMPure bead. Mix by pipetting 10x
- Wait for 15min
- Transfer to sit on magnet for 5min
- Wash twice with 200ul freshly prepared 80% EtOH
- After adding 80% EtOH (1st time), wait for 30sec before pipetting up & down 5x
- Remove all 80% EtOH
- Dry the bead for 15min
- Resuspend with 20 EB Buffer (keep the beads in the tubes!)

Methylated adaptor ligation

Components 1x rxn 11rxn Mix
dA-tailed DNA 20.00 0.00
10x Ligation Buffer 3.00 33.00
T4 DNA Ligase (6000U/ul) 1.00 11.00
TruSeq Adaptor (15uM) 1.50 0.00
H2O 4.50 49.50
Total 30.00
- Add 1.5ul of 15uM (non-diluted adaptor) TruSeq adaptor to each tube
Sample Truseq Index
HC-C1 1
HC-C2 2
HC-C3 3
HC-C4 4
HC-H1 5
HC-H2 6
HC-H3 7
HC-H4 8
HC-H5 9
NTC 10
  • Final conc. of adaptor in 30ul lgation reaction is 750nM
- Add 8.5ul of ligation reaction mix with adaptors
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 16C, 24h (no heat lid)
- Mix by gentle pulse-vortexing on PCR rack 5x
- Quickly spin down
- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
  • Before continue to bisulfite conversion, purify adaptor-ligated DNA with 2x volume AMPure beads (60ul each) and resuspend the bead with 21ul EB buffer

AMPure bead purification

- Add 60ul AMPure bead. Mix by pipetting 10x
- Wait for 15min
- Transfer to sit on magnet for 5min
- Wash twice with 200ul freshly prepared 80% EtOH
- After adding 80% EtOH (1st time), wait for 30sec before pipetting up & down 5x
- Remove all 80% EtOH
- Dry the bead for 15min
- Resuspend with 21 EB Buffer
- Wait for 2min.
- Transfer to sit on magnet for 2 min and transfer 20ul of adaptor-ligated DNA to new strip tubes (discard the beads)
- Continue to bisulfite conversion

Bisulfite conversion

- Add 130ul complete Lightning 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 (Magprep, Heated Lid)
- 98°C for 10 minutes (DNA denaturation)
- 54°C for 1 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)
- Wait for 18min
- 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 transfering to new tube
- Add 36ul 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

Amplification

TEST PCR

- Prepare PCR in total 20ul reaction with 4 ul BIS-DNA
Components Conc unit Final conc. unit Volume (ul) 10.5 rxn mix
Bis-cvt DNA 3.00 0.00
2X KAPA HiFi Uracil HotStart ReadyMix 2 X 1 X 5.00 52.50
TruS_F/R 10 uM 0.3 uM 0.30 3.15
50X SYBG 50 X 0.4 X 0.08 0.84
H2O 1.62 17.01
Total 10.00
- Aliquot 3.5ul + 1.5ul BIS-DNA
- 98C for 45sec --> [98C for 15sec -> 60C for 30sec -> 72C for 30sec] X 13X --> 72C for 1min
- Verify all PCR product in 6% TBE gel

File:ZhangLab 2 2014-12-12 20hr 24min RRBS-DZ-5ul.jpg

  • It looked like the pattern of size distribution was different from previous experiment as the size seemed to be more larger than sequencing library prepared from human tumor DNA.
  • I continued to amplified the rest of BIS-DNA in 100ul reaction with cycle number 14 cycles for sample C1, C2, H1, H3, 15 cycles for samples C4, H4, H5 and 16 cycles for sample C3
- Purified with 1X AMPure bead, elute with 32ul EB buffer
- PAGE quantify by loading 4ul
File:ZhangLab 2 2014-12-15 19hr 52min PQ SeqLib-4ul.jpg