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.