Jie:LabNotes/CpgSeq/2009-7-2: Difference between revisions
Jump to navigation
Jump to search
>Sam Chiang No edit summary |
>Sam Chiang No edit summary |
||
Line 22: | Line 22: | ||
|} | |} | ||
===Making cDNA === | |||
==Making | |||
64 65 72 73 75 | 64 65 72 73 75 | ||
Line 40: | Line 30: | ||
H2O 39.5ul 39.5ul 32.5ul 32.5 31.5 | H2O 39.5ul 39.5ul 32.5ul 32.5 31.5 | ||
37C 10min | 37C 10min | ||
Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit | |||
* Add 4 volumes of RNA binding buffer; | * Add 4 volumes of RNA binding buffer; | ||
* Transfer to Zymo-spin columns in collection tubes, spin @15k rpm for 30s; | * Transfer to Zymo-spin columns in collection tubes, spin @15k rpm for 30s; | ||
Line 46: | Line 36: | ||
* Add 10ul DNase/RNase-free water, spin @15k rpm for 30s; | * Add 10ul DNase/RNase-free water, spin @15k rpm for 30s; | ||
Reverse transcription | Reverse transcription | ||
64 | 64~75 | ||
RNA 8ul | RNA 8ul | ||
10mM dNTP 1ul | 10mM dNTP 1ul | ||
50uM dT12-18 1ul | 50uM dT12-18 1ul | ||
65C 5min -> chill on ice for 1min | 65C 5min -> chill on ice for 1min | ||
Add | Add | ||
10X RT buffer | 10X RT buffer 2ul | ||
25mM MgCl2 | 25mM MgCl2 4ul | ||
0.1M DTT 2ul | 0.1M DTT 2ul | ||
RNaseOUT | RNaseOUT 1ul | ||
Superscript III 1ul | Superscript III 1ul | ||
Incubate all tube at 50C for 50min. | Incubate all tube at 50C for 50min. | ||
Cleanup the first-strand reactions with qiaquick columns. | |||
quantify: | |||
Cleanup the first-strand reactions with qiaquick columns. | |||
Revision as of 00:06, 3 July 2009
DNA and RNA extraction from samples and ASE validation for fuzzlmethylated genes
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 |
GM12864 | 94.1ng/ul x 100ul | 1.97/0.17 | 1515.7ng/ul x 30ul | 2.1/2.2 |
GM12865 | 130.9ng/ul/ul x 100ul | 1.98/1.44 | 1515.9ng/ul x 30ul | 2.11/2.19 |
GM12872 | 217.8ng/ul x 100ul | 1.94/1.83 | 1638ng/ul x 30ul | 2.1/2.18 |
GM12873 | 82.6ng/ul x 100ul | 1.92/1.49 | 1416.4ng/ul x 30ul | 2.11/2.14 |
GM12875 | 140ng/ul x 100ul | 1.89/1.03 | 838.3ng/ul x 30ul | 2.13/2.11 |
Making cDNA
64 65 72 73 75 RNA 3ul 3ul 3ul 3ul 4ul 10X DNase I buffer 5ul 5ul 5ul 5ul 5ul RNase-Free DNase I 2.5ul 2.5ul 2.5ul 2.5 2.5 H2O 39.5ul 39.5ul 32.5ul 32.5 31.5 37C 10min Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit * Add 4 volumes of RNA binding buffer; * Transfer to Zymo-spin columns in collection tubes, spin @15k rpm for 30s; * Add 200ul RNA Wash buffer, spin @15k rpm for 30s; * Add 10ul DNase/RNase-free water, spin @15k rpm for 30s;
Reverse transcription
64~75 RNA 8ul 10mM dNTP 1ul 50uM dT12-18 1ul 65C 5min -> chill on ice for 1min Add 10X RT buffer 2ul 25mM MgCl2 4ul 0.1M DTT 2ul RNaseOUT 1ul Superscript III 1ul Incubate all tube at 50C for 50min.
Cleanup the first-strand reactions with qiaquick columns. quantify: