Noi/NOTES/2014-10-24: Difference between revisions

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>Noi
(Created page with "= Preparation of WGBS of cfDNA from plasma using KAPA Hyper Prep kit & NEXTflex Cell Free DNA-Seq kit = '''Link to calendar''' * REPEAT experimen...")
 
>Noi
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* REPEAT experiment of 2014-10-22
* REPEAT experiment of 2014-10-22
* [[Media:KAPA_Hyper_Prep_Kit_TDS.pdf|'''KAPA Hyper Prep Kit Manual''']]
* [[Media:KAPA_Hyper_Prep_Kit_TDS.pdf|'''KAPA Hyper Prep Kit Manual''']]
* I will list the procedures of KAPA Hyper Prep kit through adaptor ligation. The two experiments were performed in parallel.
* 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
* 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.
* 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 =
= KAPA Hyper Prep kit  Experiment =
'''Sample list'''<br>
'''Sample list'''<br>

Revision as of 05:33, 28 October 2014

Preparation of WGBS of cfDNA from plasma using KAPA Hyper Prep kit & NEXTflex Cell Free DNA-Seq kit

Link to calendar

  • 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 on June 2014
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.

1X AMPure bead purification