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==bisulfite conversion of PGP samples== {| border="1" cellpadding="5" cellspacing="0" align="center" |- | align="center" style="background:#f0f0f0;"|''' ''' | align="center" style="background:#f0f0f0;"|'''sample ''' |align="center" style="background:#f0f0f0;"|'''sample concentration''' | align="center" style="background:#f0f0f0;"|'''sample volumn''' | align="center" style="background:#f0f0f0;"|'''ddH2O ''' | align="center" style="background:#f0f0f0;"|'''conversion reagents''' | align="center" style="background:#f0f0f0;"|'''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== {| border="1" cellpadding="5" cellspacing="0" align="center" |- | align="center" style="background:#f0f0f0;"|''' ''' | align="center" style="background:#f0f0f0;"|'''sample ''' |align="center" style="background:#f0f0f0;"|'''sample concentration''' | align="center" style="background:#f0f0f0;"|'''10xLigase buffer''' | align="center" style="background:#f0f0f0;"|'''template+cpg92k+H2O''' | align="center" style="background:#f0f0f0;"|'''template+control probes+H2O''' | align="center" style="background:#f0f0f0;"|'''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 -> <BR>4 cycels (95C 1min -> 55C for 4h),add 2ul more SLN mix after 1st cycle-> 95C 5min -> <BR>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 -> <BR>4 cycels (95C 1min -> 55C for 4h),add 2ul more SLN mix after 1st cycle -> 95C 5min -> <BR>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)== ===PCR with AmpF/R6.3Sol and iProof=== 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. [[Image:20090524_Jan09_captured_targets_iProof_PCR_curve.png|500px]]20090524_Jan09_captured_targets_iProof_PCR_curve [[Image:20090524_Jan09_captured_targets_iProof_PCR_gel.jpg]]20090524_Jan09_captured_targets_iProof_PCR_gel ===Normalization of the PCR amplicons of captured targets(2009-05-26)=== 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. <BR>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=== 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. [[Image:20090526_amplicons after normalization of captured targets.jpg]]20090526_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.
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