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==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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