Noi/NOTES/2014-8-15

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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)