AlanFung:LabNotes/Methylome/2012-1-17: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
Line 75: | Line 75: | ||
| Total||57.14 | | Total||57.14 | ||
|} | |} | ||
*Incubate at room temperature for 30 minutes | |||
*purify the product with Ampure beads (1X) and elute in 52.5ul ddH2O | |||
==Bisulfite Conversion== | ==Bisulfite Conversion== | ||
==PCR== | ==PCR== | ||
==Size selection== | ==Size selection== |
Revision as of 18:39, 27 January 2012
GM12878 Library
Sample Preparation
- Get GM12878 samples from Noi
Took 20ul (250ng/ul) from E2 position of the sample plate, add 30ul ddh2o making final volume to 50ul for shearing
- Take 5ug of samples and prepare for shearing (Make total volume to 100ul with ddh2o)
- Covaris shearing of 5ug gDNA to ~400bp
- Contacted Yang at Kelly Frazer's lab to help me with it (so Yang says they don't have the coavris shearing machine anymore and they shear their samples at Biogem now, emailed James and see if i can get it sheared over there)
- James helped me out to shear the samples, he used the following settings
Target BP 400 Tube Micro Duty Factor 10% Peak Incident Power (w) 140 Cycle per Burst 200 Time (secs) 55 Volume 50ul Temperature (c) 7 Intensifier Yes S water level 12 E water level 6
End repair & A-tailing
End Repair
Content | Volume |
Fragmented DNA | 50 |
End Reapir buffer | 10 |
End Repair Enzyme Mix | 5 |
ddH2O | 35 |
Volume | 100 |
- incubate at 20C for 30mins
- Purify DNA with Qiaquick column
A-tailing
Content | Volume |
End repaired blunt dna | 30 |
A-tailing reaction buffer 10x | 5 |
klenow fragment | 3 |
H2o | 12 |
Total Volume | 50 |
- Incubate @ 37C for 30mins
- Qiaquick
- Keep 1ul for gel image
ligation to methylated Y adaptor
- 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
- 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
Content | Volume |
5X ligation buffer | 11.43 |
DNA Ligase | 5.71 |
Methylation Adapter Oligo | 10 |
A tailed DNA | 30 |
h2o | 0 |
Total | 57.14 |
- Incubate at room temperature for 30 minutes
- purify the product with Ampure beads (1X) and elute in 52.5ul ddH2O