Jie:LabNotes/CpgSeq/2009-5-20
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bisulfite conversion of PGP samples[edit]
sample | sample concentration | sample volumn | ddH2O | conversion reagents | conversed DNA concentration and volumn | |
09_1 | NA21687*1 062507 | 241.1ng/ul x 1 tubes | 8ul | 12ul | 130ul | 95.4ng/ul x 10ul |
09_2 | NA21781*1 102307 | 240ng/ul x 1 tubes | 8ul | 12ul | 130ul | 120.2ng/ul x 10ul |
09_3 | NA21833*1 101807 | 253.9ng/ul x 1 tube | 8ul | 12ul | 130ul | 121.5ng/ul x 10ul |
09_4 | NA21660*1 053007 | 243.2ng/ul x 1 tube | 8ul | 12ul | 130ul | 120.3ng/ul x 10ul |
09_5 | NA21731*1 101807 | 217.4ng/ul x 1 tube | 10ul | 10ul | 130ul | 127.3ng/ul x 10ul |
09_6 | NA21846*1 100407 | 186.5ng/ul x 1 tube | 11ul | 9ul | 130ul | 151.3ng/ul x 10ul |
09_7 | NA21070*1 100406 | 329.7ng/ul x 1 tube | 6ul | 14ul | 130ul | 311.2ng/ul x 10ul |
Jurkat gDNA | 140ng/ul x 3 tube | 14ul | 6ul | 130ul | 255.6ng/ul x 20ul |
- Add 900ul ddH2O, 300ul M-Dilution Buffer and 50ul M-Dissolving Buffer to a tube of CT Conversion Reagent.
- Add 130ul of CT conversion reagent to samples.
- 98C 10min -> 64C 150min -> 4c overnight.
- Add 600ul M-Binding buffer to spin columns.
- Add the converted samples to the columns, close the gap and mix by inverting several times.
- Spin at 15,000rpm for 30sec.
- Add 100ul M-Wash Buffer, spin for 30 sec, discard flow-through.
- Add 200ul M-Desulphonation Buffer, wait for 20min, spin at 15,000 rpm for 30sec.
- Add 200ul M-Wash Buffer, spin for 30 sec.
- Place the columns into 1.5ml tubes, add 10ul M-Elution Buffer. Wait for 1min, spin at 15,000rpm for 30sec.
- Measure the DNA with Nanodrop:
RNA
set up the capture reaction[edit]
sample | sample concentration | 10xLigase buffer | template+cpg92k+H2O | template+control probes+H2O | reaction condition | |
1 | bis_Jurkat | 255.6ng/ul | 1ul | 1+4ul(#1_22.3ng/ul_05/20)+4ul | 55C anneal | |
2 | bis_Jurkat | 255.6ng/ul | 1ul | 1+4ul(#1_22.3ng/ul_05/20)+4ul | 60C anneal | |
3 | bis_Jurkat | 255.6ng/ul | 1ul | 1+3ul(#2_17ng/ul_05/20)+5ul | 55C anneal | |
4 | bis_Jurkat | 255.6ng/ul | 1ul | 1+1ul(cpg30k,50ng/ul,06/13/08)+7ul | 55C anneal | |
5 | Jurkat | 142.8ng/ul | 1ul | 1+1ul(cosmic,19.3ng/ul,04/29/08)+7ul | 55C anneal | |
6 | 09_1 | 95.4ng/ul | 1ul | 2+5ul(#2_17ng/ul_05/20)+2ul | 55C anneal | |
7 | 09_7 | 311.2ng/ul | 1ul | 2+5ul(#2_17ng/ul_05/20)+2ul | 55C anneal | |
8 | negative control | 0 | 1ul | 0+4ul(#1_22.3ng/ul_05/20)+5ul | 55C anneal |
dNTP concentration 10mM(NEB) ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP: 0.5ul 0.1ul 0.1ul 0.2ul (10mM)0.1ul x30 15ul 3ul 3ul 6ul 3ul
95c 10min -> 55C 24h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 55C 4h ->
4 cycels (95C 1min -> 55C for 4h),add 2ul more SLN mix after 1st cycle-> 95C 5min ->
add 2ul Exonuclease I/III mix as soon as the temperature is lowered to 37C -> 37C 1h -> 94C 5min -> 4C hold.
tube2: 95c 10min -> 60C 24h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 60C 4h ->
4 cycels (95C 1min -> 55C for 4h),add 2ul more SLN mix after 1st cycle -> 95C 5min ->
add 2ul Exonuclease I/III mixadd 2ul Exonuclease I/III mix as soon as the temperature is lowered to 37C -> 37C 1h -> 94C 5min -> 4C hold.
PCR(05/24/2009)[edit]
PCR with AmpF/R6.3Sol and iProof[edit]
x8 Template 5ul 2X iProof Mastermix 50ul 400ul AmpF6.3SoL (10uM) 4ul 32ul AmpR6.3SoL (10uM) 4ul 32ul 50X SYBG I 0.8ul 6.4ul H2O 36.2ul 289.6ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 9 -> 72C 3 min -> 15C hold.
File:20090524 Jan09 captured targets iProof PCR curve.png20090524_Jan09_captured_targets_iProof_PCR_curve
File:20090524 Jan09 captured targets iProof PCR gel.jpg20090524_Jan09_captured_targets_iProof_PCR_gel
Normalization of the PCR amplicons of captured targets(2009-05-26)[edit]
1. Purify the No09_1 and No09_7 PCR amplicons with Qiaquick column and dissolve in 30ul EB. concentration: No09_1: 21.4ng/ul (81nM) x 30ul; No09_7: 25.3ng/ul (96nM) x 30ul. 2. Take 6ul from each sample and do the following procedure. 3. set up the control reaction: 12ul water 4ul DSN control template 2ul 10xDSN master buffer aliquot 9ul into two tubes C and E. Add 1ul DSN storage buffer into C tube. Add 1ul DSN solution into the E tube.
Overlay with oil and incubate at 65C for 10 min. Add 5ul 2xDSN stop buffer, room temperature. 4. set up the sample reactions: 5. Add 2ul 4X Hybridization buffer (2M NaCl, 200mM HEPES), 20ul mineral oil 6. 95C 2min -> 164 cycles of (97.8C 1min, -0.2C/cycle) -> 65C 90 min. 7. Preheat 2X DSN buffer for > 5min at 65C. 8. Add 8.5ul DSN buffer to each tube, 65C 10min 9. Add 0.5ul of 1U/ul DSN enzyme to each tube, 65C 25min 10. Add 17ul 2X DSN stop buffer, 95C 10min, 4C hold. 11. Purification with Qiaquick column. The concentration of both samples are ~0.1ng/ul.
PCR with AmpF/R6.3Sol and iProof[edit]
x2 Template 5ul 2X iProof Mastermix 50ul 100ul AmpF6.3SoL (10uM) 4ul 8ul AmpR6.3SoL (10uM) 4ul 8ul 50X SYBG I 0.8ul 1.6ul H2O 40.2ul 80.4ul 98C 30S -> (98C 10S -> 72C 20S) x 11 -> 72C 3 min -> 15C hold. After Qiaquick purification, use the nanodrop to calculate the concentration:
No09_1: 19.3ng/ul (73nM) x 30ul; No09_7: 23.1ng/ul (87nM) x 30ul.
File:20090526 amplicons after normalization of captured targets.jpg20090526_amplicons after normalization of captured targets
I sent 10ul of the 09_7 NA21070*1 100406 captured targets and 09_7 NA21070*1 100406 normalized captured targets to do the end sequencing and see the read abundance of probes. The sample names are labeled as:
21070*1 100406 captured targets: 96nM ; 21070*1 100406 normalized : 87nM.