Noi/NOTES/2014-8-15: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Noi
>Noi
mNo edit summary
Line 189: Line 189:
:- Elute with 42ul EB Buffer
:- Elute with 42ul EB Buffer
:- Continue to 2nd round PCR. No PAGE verification.
:- Continue to 2nd round PCR. No PAGE verification.
=== 2nd round PCR ===
=== Quick test ===
{| {{table}} border =1
| align="center" style="width:140px;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.'''
| align="center" style="width:60px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''30 rxn mix'''
|-
| Purified 1st round DNA||||||||||3.00||0.00
|-
| 5X Phusion HF buffer||5||X||1||X||2.00||60.00
|-
| dNTP mix||10||mM||0.25||mM||0.25||7.50
|-
| TruS_F/R||10||uM||0.3||uM||0.30||9.00
|-
| 50X SYBR||50||X||0.4||X||0.08||2.40
|-
| Phusion HF||2||unit/ul||||||0.10||3.00
|-
| H2O||||||||||4.27||128.10
|-
| Total||||||||||10.00||
|}
:- Aliquot 7 + 3ul DNA template
:- Mix well.
::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 8 cycles --> 72C for 2min''<br>
:- Added 2ul 6X loading dye to each strip tube & mix well
:- Load 6ul PCR/dye mix in 6% gel and run at 250 volts for 22min.
[[File:ZhangLab_2 2014-08-17 16hr 58min_test-2ndPCR_NC-8x_gel1.jpg|450px]] [[File:ZhangLab_2 2014-08-17 16hr 58min_test-2ndPCR_NC-8x_gel2.jpg|450px]]
[[File:ZhangLab_2 2014-08-17 16hr 58min_test-2ndPCR_NC-8x_gel3.jpg|450px]]

Revision as of 02:53, 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
- Aliquot 20 + 30ul BIS-DNA template
- Mix well
95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 --> 72C for 2min
- Purify with 1.25X AMPure beads
- Elute with 42ul EB Buffer
- Continue to 2nd round PCR. No PAGE verification.

2nd round PCR

Quick test

Components Conc unit Final conc. unit Volume (ul) 30 rxn mix
Purified 1st round DNA 3.00 0.00
5X Phusion HF buffer 5 X 1 X 2.00 60.00
dNTP mix 10 mM 0.25 mM 0.25 7.50
TruS_F/R 10 uM 0.3 uM 0.30 9.00
50X SYBR 50 X 0.4 X 0.08 2.40
Phusion HF 2 unit/ul 0.10 3.00
H2O 4.27 128.10
Total 10.00
- Aliquot 7 + 3ul DNA template
- Mix well.
98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 8 cycles --> 72C for 2min
- Added 2ul 6X loading dye to each strip tube & mix well
- Load 6ul PCR/dye mix in 6% gel and run at 250 volts for 22min.
File:ZhangLab 2 2014-08-17 16hr 58min test-2ndPCR NC-8x gel1.jpg File:ZhangLab 2 2014-08-17 16hr 58min test-2ndPCR NC-8x gel2.jpg

File:ZhangLab 2 2014-08-17 16hr 58min test-2ndPCR NC-8x gel3.jpg