Jie:LabNotes/CpgSeq/2009-8-10: Difference between revisions

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>Sam Chiang
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>Sam Chiang
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                                       x50  25ul    5ul      5ul    10ul    5ul
                                       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.
Qiaquick purify the amplicons and e-gel size selection.


===PCR amplification with AmpF6.3NH2/AmpR6.3NH2===
===PCR amplification with AmpF6.3NH2/AmpR6.3NH2===

Revision as of 23:27, 16 August 2009

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 amplification with AmpF6.3NH2/AmpR6.3NH2

I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample.
reaction system                                                 x4       x6  
H2O                                                  45ul      180ul     1080
2x iProof Mastermix                                  50ul      200ul     1200 
AmpF6.3NH2(10uM)                                      2ul       8ul      48 
AmpR6.3NH2(10uM)                                      2ul       8ul      48
50x SYBG I                                          0.4ul      1.6ul     9.6
template                                      1ul/each for purified amplicons
Total                                               100ul      800ul     
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 ->72C 3min.