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
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.