Noi/NOTES/2012-7-27

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Revision as of 16:00, 27 July 2012 by >Noi
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WGBS sequencing library preparation using KAPA protocol (Eppendorf)

  • Note that I used the same total volume of reaction similar to the protocol from Eppendorf. This might be slightly different from the original protocol from KAPA kit, but the total amount of enzyme and buffer are the same. I also adjust the volume of adapters in ligation step, but total volume and ratio of adapter to A-tailed DNA are the same.

Preparation of enzyme mix for End repair, A-tailing, Ligation, and Methylation adapters

  • First of all, need to check if I have enough reagents for 8 rxn
  • Since I perform library prep for exactly 8 rxn (1 column-> single pipetting), I will not prepare extra volume since I plane to set up machine to pipette all reaction mix aliquoted in the well (exactly each reaction need). This mean setting up larger volume of pipetting to make sure that all reaction mix is transfered to the DNA.
End repair 
Total reaction in        70ul		
DNA 	                 50ul
End repair reaction mix 20ul

KAPA End repair mix	       1x rxn	8x rxn mix
10X End repai buffer	       7.00	56.00
KAPA End repair enzyme	       5.00	40.00
H2O	                       8.00	64.00
==================================================
Total	               20.00   160.00
Aliquot to column #1: 20ul

20C for 30min
A-tailing
Total reaction in 30ul

A-tailing reaction mix	      1x rxn	8x rxn mix
KAPA 10X A-tailing buffer	3.00	24.00
KAPA A-tailing enzyme	        3.00	24.00
H2O	                       24.00   192.00
==================================================
Total	                       30.00   240.00
Aliquot to column #2: 30ul

30C for 30min
Adapter ligation
Total reaction in 50ul 
Ligation reaction mix 40ul
Methylation adapter   10ul

Ligation reaction mix	      1x rxn	8x rxn mix
KAPA 5X Ligation buffer	10.00	80.00
KAPA DNA ligase	         5.00	40.00
H2O	                        25.00	200.00
==================================================
Total	                        40.00	320.00
Aliquot to column #3: 40ul

Methylation adapter preparation
Alan's note: 
1. Use the methylated adapter with paired-end pcr primer pairs(PCR primer PE1.0 and PCR primer PE 2.0) provided in illumina's paired end genomic DNA sample prep kit
2. Use 10ul of methylated adapter to 5ug gDNA insert, if starting DNA amount is less than 5ug, titrate the volume of adapter reagent accordingly to maintain 10:1 ratio of DNA
Since I used 1ug of DNA, used 2ul of adapters, but dilute to final volume 10ul (This volume is larger than KAPA protocol, but the total amount is the same)

Methylation adapter	2ulx 8rxn	16.00
H2O	                 8ulx8rxn	64.00
==================================================
Total		                        80.00

25C for 15min