Noi/NOTES/2012-7-27: Difference between revisions
Jump to navigation
Jump to search
>Noi |
>Noi |
||
Line 2: | Line 2: | ||
= WGBS sequencing library preparation using KAPA protocol (Eppendorf) and epMotion = | = WGBS sequencing library preparation using KAPA protocol (Eppendorf) and epMotion = | ||
* Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-6 | * Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-6 | ||
* 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 | * 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 is 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 == | == 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 | * First of all, need to check if I have enough reagents for 8 rxn |
Revision as of 07:14, 28 July 2012
WGBS sequencing library preparation using KAPA protocol (Eppendorf) and epMotion
- Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-6
- 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 is 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
Sample list
Labeled Sample IDs 1 GM12891 2 GM12892 3 GM10861 4 GM11994 5 GM11995 6 GM18506 7 GM18507 8 GM18505
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 20C for 15min
Reagent preparation Reagents 1rxn Min volume in 30mL reservior AMPure beads 120ul ~1.1mL 20% PEG/2.5M NaCl 90+50 = 140ul ~1.4mL 80% EtOH 140x6 = 840ul ~10.0mL H2O for elution 23ul ~1.0mL
epMotion run
Report file of epMotion run: epMotion Started:4:40pm, finished: 7:30pm (~3hr for 8 samples)
- Very important, when I added AMPure bead to end-repaired DNA and mixed for ten times, I have changed liquid type to water. This should be changed back to glycerol as before since the last step, the liquid was flushed out really quick, and it might cause contamination if bubbles are formed.
- Note:
- After adapter ligation I eluted DNA with 23ul H2O, and transfered eluted DNA to 96-well plate (on the same plate, column #3)
- I checked adapter ligated DNA in 6% TBE gel (loaded 0.5 ul each, also load sheared DNA of sample #1 and #2 as control)
File:ZhangLab 2 2012-07-27 20hr 33min KAPA-adapter.jpg Labeled Sample IDs #1B = #1 before end repair #2B = #2 before end repair #1 GM12891 #2 GM12892 #3 GM10861 #4 GM11994 #5 GM11995 #6 GM18506 #7 GM18507 #8 GM18505