Noi/NOTES/2014-11-30: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Noi
(Created page with "= Preparation of WGBS of cfDNA from plasma of cancer patients using KAPA Hyper Prep kit = '''Back to calendar''' * As I mentioned before in previ...")
 
>Noi
mNo edit summary
Line 3: Line 3:
* As I mentioned before in previous experiment that the first batch of WGBS libraries from cfDNA of normal and cancer patients were generated by RRBS method without DNA fragmentation. This might lead to the incomplete of libraries as RRBS protocol uses only Klenow fragment, eco- but lack T4 DNA Pol and T4 PNK enzyme. Dr. Zhang suggested to generated WGBS libraries of cfDNA from cancer patients using KAPA Hyper Prep Kit as I did test experiment on 2014-10-24.
* As I mentioned before in previous experiment that the first batch of WGBS libraries from cfDNA of normal and cancer patients were generated by RRBS method without DNA fragmentation. This might lead to the incomplete of libraries as RRBS protocol uses only Klenow fragment, eco- but lack T4 DNA Pol and T4 PNK enzyme. Dr. Zhang suggested to generated WGBS libraries of cfDNA from cancer patients using KAPA Hyper Prep Kit as I did test experiment on 2014-10-24.
* I also increased DNA input as much as possible for each sample to be able to capture all individual unique molecule from cancer cells in the DNA pool of cfDNA.
* I also increased DNA input as much as possible for each sample to be able to capture all individual unique molecule from cancer cells in the DNA pool of cfDNA.
{| {{table}} border = 1
| align="center" style="width:80px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Sample conc. (ng/uL)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Yields (ng)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Amont left (ng)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Amount used in WGBS/ KAPAHyper'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:80px;background:#f0f0f0;"|'''H2O to 25ul'''
| align="center" style="width:80px;background:#f0f0f0;"|'''End repair/dA-tailing rxn mix'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Total (ul)'''
|-
| PC-P_2||0.852||30||25.56||22.06||20||23.47||PC-P_2||1.53||5||30.00
|-
| PC-P_3||1.360||30||40.80||37.30||20||14.71||PC-P_3||10.29||5||30.00
|-
| PC-P_4||0.340||30||10.20||6.70||6.5||19.12||PC-P_4||5.88||5||30.00
|-
| PC-P_5||0.813||30||24.39||20.89||20||24.60||PC-P_5||0.40||5||30.00
|-
| PC-P_6||0.767||30||23.01||19.51||15||19.56||PC-P_6||5.44||5||30.00
|-
| PC-P_7||0.644||30||19.32||15.82||15||23.29||PC-P_7||1.71||5||30.00
|-
| PC-P_8||0.524||30||15.72||12.22||10||19.08||PC-P_8||5.92||5||30.00
|-
| PC-P_9||0.238||30||7.14||3.64||3.5||14.71||PC-P_9||10.29||5||30.00
|-
| ||||||||||||||||||||
|-
| 6-P-1||0.200||30||6||2.50||2.5||12.50||6-P-1||12.50||5||30.00
|-
| 6-P-2||1.100||30||33||29.50||20||18.18||6-P-2||6.82||5||30.00
|-
| 6-P-3||1.570||30||47.1||43.60||20||12.74||6-P-3||12.26||5||30.00
|-
| 6-P-4||0.889||30||26.67||23.17||20||22.50||6-P-4||2.50||5||30.00
|-
| 6-P-5||0.487||30||14.61||11.11||10||20.53||6-P-5||4.47||5||30.00
|-
| 6-P-8||0.226||30||6.78||3.28||3.2||14.16||6-P-8||10.84||5||30.00
|-
| 6-P-9||0.408||30||12.24||8.74||8.5||20.83||6-P-9||4.17||5||30.00
|-
| 6-P-10||0.333||30||9.99||6.49||6.4||19.22||6-P-10||5.78||5||30.00
|-
| ||||||||||||||||||||
|-
| 7-P-1||0.692||30||20.76||17.26||15||21.68||7-P-1||3.32||5||30.00
|-
| 7-P-2||1.14||30||34.2||30.7||20||17.54||7-P-2||7.46||5||30.00
|-
| 7-P-3||0.208||30||6.24||2.74||2.5||12.02||7-P-3||12.98||5||30.00
|-
| 7-P-5||1.11||30||33.3||29.8||20||18.02||7-P-5||6.98||5||30.00
|-
| 7-P-6||0.233||30||6.99||3.49||3.4||14.59||7-P-6||10.41||5||30.00
|-
| 7-P-7||0.229||30||6.87||3.37||3.3||14.41||7-P-7||10.59||5||30.00
|-
| 7-P-8||0.167||30||5.01||1.51||1.5||8.98||7-P-8||16.02||5||30.00
|-
| 7-P-10||0.203||30||6.09||2.59||2.5||12.32||7-P-10||12.68||5||30.00
|-
| NTC||NA||NA||NA||NA||NA||NA||NTC||25.00||5||30.00
|}
== End repair & dA-tailing ==
==== End repair & dA-tailing reaction mix ====
* I prepared 5ul of End repair & dA-tailing reaction mix for each reaction
* Maximum volume for End-repair/A-tailing = 25ul
{| {{table}} border = 1
| align="center" style="width:200px;background:#f0f0f0;"|'''End repair & dA-tailing reaction mix'''
| align="center" style="width:80px;background:#f0f0f0;"|''''''
| align="center" style="width:80px;background:#f0f0f0;"|''''''
| align="center" style="width:80px;background:#f0f0f0;"|''''''
|-
| Components||Volume||half rxn||26 rxn
|-
| End repair/dA-tailing buffer ||7.00||3.50||91.00
|-
| End repair/dA-tailing enzyme mix||3.00||1.50||39.00
|-
| Total||10.00||5.00||
|}
:- Mix well
:- 20C, 30min
:- 65C, 30min,
:- Hold 4C
:- Proceed to Adaptor ligation immediately
== Adaptor ligation ==

Revision as of 10:54, 12 January 2015

Preparation of WGBS of cfDNA from plasma of cancer patients using KAPA Hyper Prep kit

Back to calendar

  • As I mentioned before in previous experiment that the first batch of WGBS libraries from cfDNA of normal and cancer patients were generated by RRBS method without DNA fragmentation. This might lead to the incomplete of libraries as RRBS protocol uses only Klenow fragment, eco- but lack T4 DNA Pol and T4 PNK enzyme. Dr. Zhang suggested to generated WGBS libraries of cfDNA from cancer patients using KAPA Hyper Prep Kit as I did test experiment on 2014-10-24.
  • I also increased DNA input as much as possible for each sample to be able to capture all individual unique molecule from cancer cells in the DNA pool of cfDNA.
Sample Sample conc. (ng/uL) Volume (ul) Yields (ng) Amont left (ng) Amount used in WGBS/ KAPAHyper Volume (ul) Sample H2O to 25ul End repair/dA-tailing rxn mix Total (ul)
PC-P_2 0.852 30 25.56 22.06 20 23.47 PC-P_2 1.53 5 30.00
PC-P_3 1.360 30 40.80 37.30 20 14.71 PC-P_3 10.29 5 30.00
PC-P_4 0.340 30 10.20 6.70 6.5 19.12 PC-P_4 5.88 5 30.00
PC-P_5 0.813 30 24.39 20.89 20 24.60 PC-P_5 0.40 5 30.00
PC-P_6 0.767 30 23.01 19.51 15 19.56 PC-P_6 5.44 5 30.00
PC-P_7 0.644 30 19.32 15.82 15 23.29 PC-P_7 1.71 5 30.00
PC-P_8 0.524 30 15.72 12.22 10 19.08 PC-P_8 5.92 5 30.00
PC-P_9 0.238 30 7.14 3.64 3.5 14.71 PC-P_9 10.29 5 30.00
6-P-1 0.200 30 6 2.50 2.5 12.50 6-P-1 12.50 5 30.00
6-P-2 1.100 30 33 29.50 20 18.18 6-P-2 6.82 5 30.00
6-P-3 1.570 30 47.1 43.60 20 12.74 6-P-3 12.26 5 30.00
6-P-4 0.889 30 26.67 23.17 20 22.50 6-P-4 2.50 5 30.00
6-P-5 0.487 30 14.61 11.11 10 20.53 6-P-5 4.47 5 30.00
6-P-8 0.226 30 6.78 3.28 3.2 14.16 6-P-8 10.84 5 30.00
6-P-9 0.408 30 12.24 8.74 8.5 20.83 6-P-9 4.17 5 30.00
6-P-10 0.333 30 9.99 6.49 6.4 19.22 6-P-10 5.78 5 30.00
7-P-1 0.692 30 20.76 17.26 15 21.68 7-P-1 3.32 5 30.00
7-P-2 1.14 30 34.2 30.7 20 17.54 7-P-2 7.46 5 30.00
7-P-3 0.208 30 6.24 2.74 2.5 12.02 7-P-3 12.98 5 30.00
7-P-5 1.11 30 33.3 29.8 20 18.02 7-P-5 6.98 5 30.00
7-P-6 0.233 30 6.99 3.49 3.4 14.59 7-P-6 10.41 5 30.00
7-P-7 0.229 30 6.87 3.37 3.3 14.41 7-P-7 10.59 5 30.00
7-P-8 0.167 30 5.01 1.51 1.5 8.98 7-P-8 16.02 5 30.00
7-P-10 0.203 30 6.09 2.59 2.5 12.32 7-P-10 12.68 5 30.00
NTC NA NA NA NA NA NA NTC 25.00 5 30.00

End repair & dA-tailing

End repair & dA-tailing reaction mix

  • I prepared 5ul of End repair & dA-tailing reaction mix for each reaction
  • Maximum volume for End-repair/A-tailing = 25ul
End repair & dA-tailing reaction mix ' ' '
Components Volume half rxn 26 rxn
End repair/dA-tailing buffer 7.00 3.50 91.00
End repair/dA-tailing enzyme mix 3.00 1.50 39.00
Total 10.00 5.00
- Mix well
- 20C, 30min
- 65C, 30min,
- Hold 4C
- Proceed to Adaptor ligation immediately

Adaptor ligation