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.