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==qPCR amplification== *Only use half of amount (10uL)for qPCR amplification. *Comparing primers (primer from Alice vs. primers I ordered) Master mix - Sam's primer mix - for L1, L3, L5, L7, N5 5.5 rxn H2O 35.6 195.8 Template (ligation product) 10.0 - Primer mix (f+r) (10uM) 4.0 22.0 SYBR 50X 0.4 2.2 2X iProof enzyme mix 50.0 275.0 --------------------------------------------- 100.0 495.0/5.5=90 --- 10uL template Master mix - Alice's primers - for L2, L4, L6, L8, N6 5.5 rxn H2O 31.6 173.8 Template (ligation product) 10.0 - Primer f(10uM) 4.0 22.0 Primer r(10uM) 4.0 22.0 SYBR 50X 0.4 2.2 2X iProof enzyme mix 50.0 275.0 --------------------------------------------- 100.0 495.0/5.5=90 --- 10uL template *Using the new program modifed based on Dr.Zhang's program (posted on 9-21-'09) **Use 5 cycles for the first annealing temp(64). **Use 16 cycles for the second annealing temp(72C). Line1: 98C 30sec Line2: 98C 10sec Line3: 64C 20sec Line4: 72C 20sec Line5: Read plate Line6: Goto line 5 for 4 more times Line7: 98C 10sec Line8: 72C 20sec Line9: Read plate Line10: Goto line 7 for 15 more times Line11: 72C 5min Line12: 15C forever END *Always terminate/pause the reaction at annealing step (72C). *Usually the reaction will be close to saturation status around 7~8 cycles. Press the skip buttom to jump to final annealing step when it's closing to saturation stage. *Check the qPCR product (3uL) on TBE gel to evaluate the library results. [[Image:sam102709-TBE Solexa lib qPCR results-2.jpg|400px]] *The library sizes are as expected. The missed L6 sample was shown on the right size on N6 sample (The additional cutting for L6 sample) **270bp + Y-adaptor = 370bp; 130bp + Y-adaptor = 230bp *The primer mix I made showed a stronger primer remains (~25bp) on the gel. *Little smear were shown on L4 and L8 samples which may be caused by a over amplification.
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