Noi/NOTES/2014-10-24: Difference between revisions
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* I used 1:20 diluted Truseq adaptor prepared in June 2014 | * I used 1:20 diluted Truseq adaptor prepared in June 2014 | ||
<u>'''KAPA Hyper Prep'''</u> | |||
:{| {{table}} border = 1 | :{| {{table}} border = 1 | ||
| align="center" style="width:80px;background:#f0f0f0;"|'''Sample IDs''' | | align="center" style="width:80px;background:#f0f0f0;"|'''Sample IDs''' |
Revision as of 05:36, 28 October 2014
Preparation of WGBS of cfDNA from plasma using KAPA Hyper Prep kit & NEXTflex Cell Free DNA-Seq kit
- REPEAT experiment of 2014-10-22
- KAPA Hyper Prep Kit Manual
- The two experiments were performed in parallel. I will list the procedures of KAPA Hyper Prep kit through adaptor ligation.
- I then will list the procedure of NEXTflex Cell Free DNA-Seq kit
- The down stream procedures, including bead purification of adaptor-ligated DNA, bisulfite conversion and amplification, were performed together and will be listed in the last part of this lab note.
KAPA Hyper Prep kit Experiment
Sample list
No. Sample IDs Conc. (ng/ul) #1 NC-2 0.325 #2 NC-3 0.538 #3 6P-2 1.10 #4 6P-3 1.57
- Note: I will reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally.
- KAPA Hyper Prep reaction set up (exactly in the instruction):
- - 50ul of fragmented DNA
- - 7ul of End repair & dA-tailing buffer
- - 3ul of End repair & dA-tailing enzyme mix
- - Total reaction: 60ul
End repair & dA-tailing
End repair & dA-tailing reaction mix
Components | Volume | half rxn | 5.2x rxn |
End repair/dA-tailing buffer | 7.00 | 3.50 | 18.20 |
End repair/dA-tailing enzyme mix | 3.00 | 1.50 | 7.80 |
Total | 10.00 | 5.00 |
Reaction set up
Sample IDs | Conc. (ng/ul) | Volume for 1ng (ul) | End repair/dA-tailing rxn mix | H2O (ul) | Total (ul) |
NC-2 | 0.33 | 3.08 | 5.00 | 21.92 | 30.00 |
NC-3 | 0.54 | 1.86 | 5.00 | 23.14 | 30.00 |
6P-2 | 1.10 | 0.91 | 5.00 | 24.09 | 30.00 |
6P-3 | 1.57 | 0.64 | 5.00 | 24.36 | 30.00 |
NTC | 0.00 | 0.00 | 5.00 | 25.00 | 30.00 |
- - Mix the reaction mix well
- - 20C, 30min
- - 65C, 30min,
- - Hold 4C
- - Proceed to Adaptor ligation immediately
Adaptor ligation
Adaptor ligation reaction mix
Components | Volume | half rxn | 5.2x rxn |
Ligation buffer | 30.00 | 15.00 | 78.00 |
DNA ligase | 10.00 | 5.00 | 26.00 |
H2O | 8.00 | 4.00 | 20.80 |
Total | 48.00 | 24.00 |
Adaptor ligation reaction set up
Components | Volume (ul) |
End repair & dA-tailing rxn product | 30.00 |
1:20 diluted adaptor (750nM) | 1.00 |
Adaptor ligation rxn mix | 24.00 |
Total | 55.00 |
- I used 1:20 diluted Truseq adaptor prepared in June 2014
KAPA Hyper Prep
Sample IDs Index NC-2 11 NC-3 12 6P-2 21 6P-3 22 NTC 11
- - Add 1ul of 1:20 diluted M-adaptor
- - Add 24ul of adaptor ligation reaction mix
- - Mix well
- - 20C, 15min
- - Continue to SPRI clean up immediately
- Final conc. of adaptor in 55ul reaction is ~13.8nM, which is very close to 14nM recommended by the kit.