Jie:LabNotes/CpgSeq/2009-8-10

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DNA and RNA extraction from iPS_DF6.9h_P28_17July09 and capture with cpg30k and cpg97k[edit]

DNA and RNA extraction with Qiagen Allprep DNA/RNA Mini kit[edit]

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[edit]

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[edit]

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[edit]

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.
mix the cpg30k and cpg30kV2.1 with 1:1 ratio; mix the cpg97k_A and B with 1:1 ratio.

Digestion with MmeI[edit]

                                                  x2 
Total                                40ul                  
PCR                                  15ul         
10X NEBuffer 4                       4ul          8           
1mM SAM(fresh)                       8ul         16             
2U/ul Mme I                          10ul        20               
ddH2O                                 3ul         6  

1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
37C 2h. MinEulte column purification. Elute in 12ul EB.

File:20090818 DF6.9 P28 MmeI digestion.jpg

Quaquick purification and quantify with nanodrop:
DF6.9_cpg30K: 600ng;
DF6.9_cpg97K: 660ng.