Noi/NOTES/2014-6-20: Difference between revisions
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:- Added 10ul 2X loading dye to each strip tube & mix well | :- Added 10ul 2X loading dye to each strip tube & mix well | ||
:- Load 10ul PCR/dye mix in 12% gel and run at 250 volts for 40min | :- Load 10ul PCR/dye mix in 12% gel and run at 250 volts for 40min | ||
[[File:ZhangLab_2 2014-06-23 11hr 45min_1ngRRBS_2nd_TT-10X.jpg| 500px]] [[File:ZhangLab_2 2014-06-23 11hr 45min_1ngRRBS_2nd_TEST-12X.jpg| 500px]] | |||
[[File:ZhangLab_2 2014-06-23 11hr 45min_1ngRRBS_2nd_TEST-14X.jpg| 500px]] [[File:ZhangLab_2 2014-06-23 11hr 45min_1ngRRBS_2nd_TEST-16X.jpg| 500px]] |
Revision as of 02:53, 24 June 2014
Preparation of low-input (1ng) RRBS sequencing libraries of cell-free DNA (plasma samples) from 10 pancreastic cancer patients and 10 normal control
Sample info
1) MspI digestion
- Note that I had condition of MspI digestion of this experiment different from the low-input experiment (5ng) on 06-03-2014 since I used 2ul of MspI in 18ul reaction. I need to increase volume of of Klenow exo- reaction and ligation to 25 and 30ul, respectively to reduce concentration of glycerol in the reaction as there is no purification steps prior to bisulfite conversion.
- For the future, I do not need to use 2ul of MspI as 1ul showed similar activity to 2ul based on agarose gel analysis. I can use the same condition as experiment on 06-03-2014
- I spike in 1% of unmeth lambda DNA. This mean that I included 10pg of lambda DNA for 1ng input DNA.
MspI digestion set up
1.1) PC-P samples
# | Sample | Original conc. (ng/ul) | Volume for 1ng (ul) | H2O to 10ul | MspI Mix | Total | Code |
1 | PC-P_1 | 0.473 | 2.11 | 7.89 | 8.00 | 18.00 | P_1 |
2 | PC-P_2 | 0.852 | 1.17 | 8.83 | 8.00 | 18.00 | P_2 |
3 | PC-P_3 | 1.360 | 0.74 | 9.26 | 8.00 | 18.00 | P_3 |
4 | PC-P_4 | 0.340 | 2.94 | 7.06 | 8.00 | 18.00 | P_4 |
5 | PC-P_5 | 0.813 | 1.23 | 8.77 | 8.00 | 18.00 | P_5 |
6 | PC-P_6 | 0.767 | 1.30 | 8.70 | 8.00 | 18.00 | P_6 |
7 | PC-P_7 | 0.644 | 1.55 | 8.45 | 8.00 | 18.00 | P_7 |
8 | PC-P_8 | 0.524 | 1.91 | 8.09 | 8.00 | 18.00 | P_8 |
9 | PC-P_9 | 0.238 | 4.20 | 5.80 | 8.00 | 18.00 | P_9 |
10 | PC-P_10 | 0.370 | 2.70 | 7.30 | 8.00 | 18.00 | P_10 |
11 | NTC_1 | 0.000 | 0.00 | 10.00 | 8.00 | 18.00 | P_11 |
12 | NTC_2 | 0.000 | 0.00 | 10.00 | 8.00 | 18.00 | P_12 |
1.2) NC-P samples
# | Sample | Original conc. (ng/ul) | Volume for 1ng (ul) | H2O to 10ul | MspI Mix | Total | Code |
1 | NC-1 | 0.293 | 3.41 | 6.59 | 8.00 | 18.00 | N_1 |
2 | NC-2 | 0.325 | 3.08 | 6.92 | 8.00 | 18.00 | N_2 |
3 | NC-3 | 0.538 | 1.86 | 8.14 | 8.00 | 18.00 | N_3 |
4 | NC-5 | 0.156 | 6.41 | 3.59 | 8.00 | 18.00 | N_5 |
5 | NC-6 | 0.148 | 6.76 | 3.24 | 8.00 | 18.00 | N_6 |
6 | NC-7 | 0.245 | 4.08 | 5.92 | 8.00 | 18.00 | N_7 |
7 | NC-8 | 0.141 | 7.09 | 2.91 | 8.00 | 18.00 | N_8 |
8 | NC-9 | 0.171 | 5.85 | 4.15 | 8.00 | 18.00 | N_9 |
9 | NC-10 | 0.134 | 7.46 | 2.54 | 8.00 | 18.00 | N_10 |
10 | NC-11 | 0.152 | 6.58 | 3.42 | 8.00 | 18.00 | N_11 |
11 | NTC_1 | 0.000 | 0.00 | 10.00 | 8.00 | 18.00 | N_12 |
12 | NTC_2 | 0.000 | 0.00 | 10.00 | 8.00 | 18.00 | N_13 |
MspI Reaction Mix
Components | 1x rxn | 26x rxn |
MspI | 2.00 | 52.00 |
10x Tango Buffer | 2.00 | 52.00 |
12pg/ul unmeth lambda DNA | 0.83 | 21.67 |
H2O | 3.17 | 82.33 |
Total | 8.00 |
- - Add H2O to each tube
- - Add 1ng of DNA to each tube
- - Add 8ul 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
Prep
- I increased reaction volume to 25ul as reason above
Components | 1x rxn | 28x rxn |
MspI-digested DNA | 18.00 | 0.00 |
10X Tango Buffer | 0.50 | 14.00 |
Klenow, exo- | 1.00 | 28.00 |
dA:dC:dG | 1.00 | 28.00 |
H2O | 4.50 | 126.00 |
Total | 25.00 | 196.00 |
- - Aliquot 24.50 of Klwnow, exo- and dA:dC:dG reaction mix to 8-tube strip
- - Add 7ul of Klwnow, exo- and dA:dC:dG reaction mix to each tube of MspI digested DNA. Pipette few times
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- - 75C, 10min --> hold at 4C (heat lid on)
- No AMPure bead purification
3) Methylated adaptor ligation
- Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 30ul
Ligation reaction set up
Prep
- Dilute 1:20 TruSeq adaptor by adding 2ul of TruSeq adaptor to 40ul H2O
Ligation reaction Mix
Components | 1x rxn | 26x rxn |
dA-tailed DNA | 25.00 | 0.00 |
10X Tango buffer | 0.50 | 13.00 |
HC T4 DNA ligase (30units/ul) | 1.00 | 26.00 |
10mM ATP | 1.50 | 39.00 |
H2O | 1.00 | 26.00 |
Total | 29.00 |
- - Aliquot 13ul of ligation reaction mix to 8-tube strip
PC-P samples
Sample Index PC_P_1 1 PC_P_2 2 PC_P_3 4 PC_P_4 5 PC_P_5 6 PC_P_6 7 PC_P_7 8 PC_P_8 10 PC_P_9 11 PC_P_10 12 NTC_1 11 NTC_2 12
NC-P samples
Sample Index NC-1 13 NC-2 14 NC-3 15 NC-5 16 NC-6 27 NC-7 18 NC-8 19 NC-9 20 NC-10 21 NC-11 22 NTC_1 21 NTC_2 22
- - Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
- - Add 4ul of ligation reaction mix
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 16C, 30h (no heat lid) --> I had no time to come back to continue to next step. I incubate longer.
- - Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
- - Continue to bisulfite conversion
4) Bisulfite conversion
- Performed bisulfite conversion using the same procedure following manufacturer's instruction and elute with 30ul Elution buffer.
Prep
- - Prepare 2 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
- - Mix well and spin down the tube
- - Put on thermomixer at 37C for 15min, 1000rpm
- - 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)
- - 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 30ul 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
1st round PCR (12X cycles
Components | Conc | unit | Final conc./amount | unit | Volume (ul) | 25 rxn mix |
Bis-cvt DNA | 30.00 | 0.00 | ||||
10X Reaction buffer | 10 | X | 1 | X | 5.00 | 125.00 |
dNTP mix | 10 | mM | 0.25 | mM | 1.25 | 31.25 |
TruS_F/R | 10 | uM | 0.3 | uM | 1.50 | 37.50 |
50X SYBG | 50 | X | 0.8 | X | 0.80 | 20.00 |
PfuTurbo Cx | 2.5 | Unit/ul | 1 | unit | 1.00 | 25.00 |
H2O | 10.45 | 261.25 | ||||
Total | 50.00 |
- - Aliquot 20 + 30ul BIS-DNA template
- - Mix well
- 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 15min, the rest steps are the same as previous experiment)
- - Elute with 40ul EB Buffer
1st round PCR (12X cycles)
OPTIMIZE PCR cycle number
- I optimize PCR cycle number between 10-16 cycles by doing PCR in small volume (~10ul) and stopped reaction at 10X, 12X, 14X, and 16X cycles.
- Then analyze in 12% TBE gel
- Select the optimum cycle number to amplify the rest of 1st round PCR
PC-P smaple
Components | Conc | unit | Final conc. | unit | Volume (ul) | 13 rxn mix |
Purified 1st round DNA | 12.00 | 0.00 | ||||
5X Phusion HF buffer | 5 | X | 1 | X | 8.00 | 104.00 |
dNTP mix | 10 | mM | 0.25 | mM | 1.00 | 13.00 |
TruS_F/R | 10 | uM | 0.3 | uM | 1.20 | 15.60 |
50X SYBR | 50 | X | 0.4 | X | 0.32 | 4.16 |
Phusion HF | 2 | unit/ul | 0.80 | 10.40 | ||
H2O | 16.68 | 216.84 | ||||
Total | 40.00 |
- - Aliquot 28 + 12ul DNA template
- - Mix well. Split 4X of 8ul
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X [10X, 12X, 14X, 16X] cycles --> 72C for 2min
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X [10X, 12X, 14X, 16X] cycles --> 72C for 2min
- - Added 10ul 2X loading dye to each strip tube & mix well
- - Load 10ul PCR/dye mix in 12% gel and run at 250 volts for 40min
File:ZhangLab 2 2014-06-23 11hr 45min 1ngRRBS 2nd TT-10X.jpg File:ZhangLab 2 2014-06-23 11hr 45min 1ngRRBS 2nd TEST-12X.jpg File:ZhangLab 2 2014-06-23 11hr 45min 1ngRRBS 2nd TEST-14X.jpg File:ZhangLab 2 2014-06-23 11hr 45min 1ngRRBS 2nd TEST-16X.jpg