Jie:LabNotes/CpgSeq/2010-1-22

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capture of mouse ES cells from Y Shi in Harvard[edit]

generation of cpgmm9A[edit]

PCR reaction                                            x 48     x96   
H2O                                   49ul             2352ul   4704
EconoTaq 2xMastermix                  50ul             2400ul   4800
Ap1V6IU       (100uM)                0.3ul             14.4ul   28.8   
Ap2V6         (100uM)                0.3ul             14.4ul   28.8
SYBR  green (50x)                    0.4ul             19.2ul   38.4
template(cpgmm9A,10uM)              0.05ul              2.4ul    4.8
Total                                100ul             4800ul   9600
Reaction program: 94c 2min -> (94c 30sec -> 60C 1min -> 72C 45sec)x  13ycles -> 72C 5min -> 15C hold.
I did the ethanol precipitation of PCR amplicon, Lambda exonuclease digestion, UESR and DpnII digestion (with DpnII-oligo_v6). The yield of probe is
38ng/ul x 20ul

bisulfite conversion of samples[edit]

Sample label on the tube from Harvard:
ct1 mES gDNA 11.20.09: 120ng/ul
Tet1-KD mES gDNA 11.20.09: 120ng/ul
I rechecked the concentration with nanodrop:
ct1 mES gDNA: 185.4ng/ul
Tet1-KD mES gDNA: 163.7ng/ul
I used 10ul of each sample, added 10ul ddH2O and 130ul conversion reagents to do the bisulfite conversion. I did 2 tubes for each sample. Yield: 
ct1 mES: 213.4ng/ul x 13ul;
Tet1 mES: 194.4ng/ul x 13ul.

capture of samples[edit]

 10X AmpLigase buffer                 1uL            
 Cmm09A Probe (38ng/ul)               2uL        
 H2O                                  2ul         
 ct1 mES: 213.4ng/ul                  2ul
 Tet1 mES: 194.4ng/ul                 2ul

95c 10min -> 60C 14h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 60C 8h -> add 2ul more SLN mix -> 60C 14h-> 95C 5min -> add 2ul Exonuclease I/III mix-> 37C 2h -> 94C 5min -> 4C hold.

PCR[edit]

I did 400ul for each sample.
2X iProof Mastermix     50ul      
AmpF6.3NH2 (10uM)        2ul       
AmpR6.3NH2 (10uM)        2ul          
50X SYBG I             0.4ul      
H2O                   42.6ul    
template                 3ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 17 ->72C 3 min -> 15C hold. 
File:20100127 mouse cmm09A capture.jpg20100127_mouse cmm09A capture
I qiaquick purified the amplicons. Sent 200ul product (400ng) for Covaris shearing.