Jie:LabNotes/CpgSeq/2009-8-10

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Revision as of 21:38, 14 August 2009 by >Sam Chiang
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DNA and RNA extraction from iPS_DF6.9h_P28_17July09 and capture with cpg30k and cpg97k

DNA and RNA extraction with Qiagen Allprep DNA/RNA Mini kit

sample DNA concentration DNA 260/280;260/230 RNA concentration RNA 260/280;260/230
iPS_DF6.9h_P28_17July09 258.9ng/ul x 100ul 2/1.68 3248.2ng/ul x 30ul 1.96/1.96


bisulfite conversion of gDNA

No sample sample concentration sample volumn ddH2O conversion reagents conversed DNA concentration and volumn
iPS_DF6.9h_P28_17July09 258.90ng/ul x 3 tube 7ul 13ul 130ul 209.7ng/ul x 20ul


capture the samples with two cpg97k subsets and 4 cpg30k subsets

The probe set was prepared on 2009-08-10

No sample sample concentration 10xLigase buffer template+cpg97k_A(60ng/ul_08/10) vol+suppress oligo+H2O template+cpg97k_B(60ng/ul_08/10) vol+suppressor oligo+H2O
iPS_DF6.9h_P28_17July09 209.7ng/ul 1ul 2+1.5ul+1ul+4.5ul 2+1.5ul+1ul+4.5ul


No sample sample concentration 10xLigase buffer template+cpg30k(5ng/ul)+suppressor(8nM each)vol+H2O template+v2.1(21ng/ul) vol+H2O template+v2.2(21ng/ul) vol+H2O template+v2.3(20ng/ul) vol+H2O
09_8 DF6-9-9_P16(4) 209.7ng/ul x 10ul 1ul 2+6+1 2+1.5+6.5 3+1.5+4.5 2+1.5+5.5 2+5ul+2ul
dNTP concentration 10mM(NEB) 
                                          H2O  Ligase Buffer Ligase  Amplitaq  dNTP           
2U/ul Stoffel; 0.5U/ul AmpLigase; 1mM dNTP: 0.5ul    0.1ul    0.1ul   0.2ul    (10mM)0.1ul 
                                      x50  25ul     5ul      5ul     10ul     5ul

PCR

Template                15ul        x12
2X iProof Mastermix     50ul      600ul
AmpF6.3SoL (10uM)        4ul       48ul
AmpR6.3SoL (10uM)        4ul       48ul   
50X SYBG I             0.4ul      4.8ul
H2O                   25.6ul    307.2ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 ->(98C 10S -> 72C 20S) x 8 ->72C 3 min -> 15C hold.