Noi/NOTES/2014-8-15: Difference between revisions
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>Noi (Created page with "= Preparation of WGBS libraries of small fragment DNA in normal control plasma samples (no DNA fragmentation step, with dAdGdCdT for end repair) = {| {{table}} border =1 | ali...") |
>Noi mNo edit summary |
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| Total||24.00|| | | Total||24.00|| | ||
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{| {{table}} border = 1 | {| {{table}} border = 1 | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
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* Repeat all procedure exactly the same as previous experiment on 2014-08-03 | * Repeat all procedure exactly the same as previous experiment on 2014-08-03 | ||
=== 1st round PCR (fix 12 cycles) === | === 1st round PCR (fix 12 cycles) === | ||
{| {{table}} border =1 | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc./amount''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | |||
|- | |||
| Bis-cvt DNA||||||||||30.00 | |||
|- | |||
| 10X Reaction buffer||10||X||1||X||5.00 | |||
|- | |||
| dNTP mix||10||mM||0.25||mM||1.25 | |||
|- | |||
| TruS_F/R||10||uM||0.3||uM||1.50 | |||
|- | |||
| 50X SYBG||50||X||0.4||X||0.40 | |||
|- | |||
| PfuTurbo Cx||2.5||Unit/ul||1||unit||1.00 | |||
|- | |||
| H2O||||||||||10.85 | |||
|- | |||
| Total ||||||||||50.00 | |||
|} |
Revision as of 02:40, 14 September 2014
Preparation of WGBS libraries of small fragment DNA in normal control plasma samples (no DNA fragmentation step, with dAdGdCdT for end repair)
Zhang lab ID | Conc. (ng/ul) | Volume for 1ng | H2O | # in strip | Exp ID | Klenow,exo-:dAdCdGdT Mix | Total |
NC-1 | 0.293 | 3.41 | 6.59 | #1_1 | wNC-1 | 10.00 | 20.00 |
NC-2 | 0.325 | 3.08 | 6.92 | #1_2 | wNC-2 | 10.00 | 20.00 |
NC-3 | 0.538 | 1.86 | 8.14 | #1_3 | wNC-3 | 10.00 | 20.00 |
NC-5 | 0.156 | 6.41 | 3.59 | #1_4 | wNC-5 | 10.00 | 20.00 |
NC-6 | 0.148 | 6.76 | 3.24 | #1_5 | wNC-6 | 10.00 | 20.00 |
NC-7 | 0.245 | 4.08 | 5.92 | #1_6 | wNC-7 | 10.00 | 20.00 |
NC-8 | 0.141 | 7.09 | 2.91 | #1_7 | wNC-8 | 10.00 | 20.00 |
NC-9 | 0.171 | 5.85 | 4.15 | #1_8 | wNC-9 | 10.00 | 20.00 |
NC-12 | 0.163 | 6.13 | 3.87 | #2_1 | wNC-12 | 10.00 | 20.00 |
NC-13 | 0.168 | 5.95 | 4.05 | #2_2 | wNC-13 | 10.00 | 20.00 |
NC-14 | 0.157 | 6.37 | 3.63 | #2_3 | wNC-14 | 10.00 | 20.00 |
NC-15 | 0.118 | 8.47 | 1.53 | #2_4 | wNC-15 | 10.00 | 20.00 |
NC-16 | 0.140 | 7.14 | 2.86 | #2_5 | wNC-16 | 10.00 | 20.00 |
NC-17 | 0.126 | 7.94 | 2.06 | #2_6 | wNC-17 | 10.00 | 20.00 |
NC-18 | 0.189 | 5.29 | 4.71 | #2_7 | wNC-18 | 10.00 | 20.00 |
NC-19 | 0.105 | 9.52 | 0.48 | #2_8 | wNC-19 | 10.00 | 20.00 |
NC-20 | 0.128 | 7.81 | 2.19 | #3_1 | wNC-20 | 10.00 | 20.00 |
NC-21 | 0.205 | 4.88 | 5.12 | #3_2 | wNC-21 | 10.00 | 20.00 |
NC-22 | 0.180 | 5.56 | 4.44 | #3_3 | wNC-22 | 10.00 | 20.00 |
NC-23 | 0.153 | 6.54 | 3.46 | #3_4 | wNC-23 | 10.00 | 20.00 |
NC-24 | 0.140 | 7.14 | 2.86 | #3_5 | wNC-24 | 10.00 | 20.00 |
NC-27 | 0.124 | 8.06 | 1.94 | #3_6 | wNC-27 | 10.00 | 20.00 |
NC-29 | 0.109 | 9.17 | 0.83 | #3_7 | wNC-29 | 10.00 | 20.00 |
NC-30 | 0.099 | 10.00 | 0.00 | #3_8 | wNC-30 | 10.00 | 20.00 |
NTC_1 | 0 | 0.00 | 10.00 | #4_1 | 10.00 | 20.00 | |
NTC_2 | 0 | 0.00 | 10.00 | #4_2 | 10.00 | 20.00 | |
NC-3 | 0.538 | 1.86 | 8.14 | #4_3 | wNC-3t | 10.00 | 20.00 |
NC-4 | 0.848 | 1.18 | 8.82 | #4_4 | wNC-4t | 10.00 | 20.00 |
- The last two samples, NC-3 and NC-4 were used to tested for T4 DNA ligase HC that has been delivered during weekend (Saturday) and was left at RT until Monday. I got the new tube from Thermoscientific for replacement. All samples except the last two were prepared with the new tube of T4 DNA ligase HC.
- Prepare new tube of dA:dC:dG:dT (20:2:2:2 mM)
Components | Final conc. (nM) | Volume (ul) |
100mM dATP | 20 | 100 |
100mM dCTP | 2 | 10 |
100mM dGTC | 2 | 10 |
100mM dTTC | 2 | 10 |
H2O | 370 |
Klenow exo- and dA:dG:dC reaction mix
Components | 1x rxn | 30x rxn |
10X Tango buffer | 2.00 | 60.00 |
dA:dC:dG:dT (20:2:2:2mM) | 1.00 | 30.00 |
Klenow fragment, exo- (5U/ul) | 1.00 | 30.00 |
H2O | 6.00 | 180.00 |
Total | 10.00 |
- Aliquot 32 of Klwnow, exo- and dA:dC:dG:dT reaction mix to 8-tube strip
- - Add H2O to each tube
- - Add 1ng of DNA to each tube
- - Add 10ul of Klwnow, exo- and dA:dC:dG reaction mix to each tube of DNA with multichannel pipette. 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
Ligation reaction mix
Components | 1x rxn | 28x rxn |
dA-tailed DNA | 20.00 | 0.00 |
10X Tango buffer | 0.50 | 14.00 |
HC T4 DNA ligase (30units/ul) | 1.00 | 28.00 |
10mM ATP | 1.25 | 35.00 |
H2O | 1.25 | 35.00 |
Total | 24.00 |
Components | 1x rxn | 2x rxn |
dA-tailed DNA | 20.00 | 0.00 |
10X Tango buffer | 0.50 | 1.00 |
THAWED HC T4 DNA ligase (30units/ul) | 1.00 | 2.00 |
10mM ATP | 1.25 | 2.50 |
H2O | 1.25 | 2.50 |
Total | 24.00 |
- - Aliquot 13ul of ligation reaction mix to 8-tube strip
- - 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 with multichannel pipette
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 16C, 20h (no heat lid)
- - Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
- - Continue to bisulfite conversion
Bisulfite conversion
Bisulfite conversion procedures
Amplification
- Repeat all procedure exactly the same as previous experiment on 2014-08-03
1st round PCR (fix 12 cycles)
Components | Conc | unit | Final conc./amount | unit | Volume (ul) |
Bis-cvt DNA | 30.00 | ||||
10X Reaction buffer | 10 | X | 1 | X | 5.00 |
dNTP mix | 10 | mM | 0.25 | mM | 1.25 |
TruS_F/R | 10 | uM | 0.3 | uM | 1.50 |
50X SYBG | 50 | X | 0.4 | X | 0.40 |
PfuTurbo Cx | 2.5 | Unit/ul | 1 | unit | 1.00 |
H2O | 10.85 | ||||
Total | 50.00 |