Gene Synthesis - Assembly PCR
1) Preparation of oligo mix. Add 500 l H2O to each lyophilized oligos. Calculate the concentration of each oligos. Add amount of oligo stock solutions to make an oligo mixture containing 2 pmol/l each oligo.
2) Remove a tube of Pfu reaction mix (for 20 l rnx) from –20 C freezer. Add 1.4 (2.3) l of water and 1 (0.1) l of oligo mix. Then add 0.2-0.4 l of Pfu DNA polymerase (2.5U/l, Stratagene or Fermentas).
3) Assembly PCR program:
Cycle temperature time
1 95 C 2 min
30 95 C 20 s
54 C 25 s
72 C 25 s
1 72 C 5 min
4) Remove two tube of Pfu reaction mix (for 20 l rnx) from –20 C freezer. Add 1.2 l of forward and reverse primers and add 0.5 l PCR reaction mix from step 3) to tube 1 and 1.0 l to tube 2. Then add 0.3 l of Pfu DNA polymerase (2.5U/l, Stratagene or Fermentas).
猎结构5) PCR program for gene amplification: Cycle temperature time
1 95 C 2 min
35 95 C 20 s
54 C 25 s
72 C 30-50 s
f型钢
1 72 C 5 min
Experimental
1) January 18, 2003 Assembly on codon-optimized cytochrome P450 monooxygenase pc-1 gene (Yadav) (990 bp)
Oligo 1 – 13, 90 mer; oligo 14, 70 mer.
Experiment 1: oligo concentration 1 or 4.8 pmole each. 58C touchdown. 2 PCR, No amplification, no smearing.
Experiment 2: oligo concentration 1 or 4.8 pmole each. 52C touchdown. 2 PCR, No amplification, no smearing.
Experiment 3: two separate reactions: oligo 1-6 and 7-14. oligo 4.8 pmole each. 52C touchdown. 2 PCR, 1 or 5 l each of 1-6 and 7-14, No amplification, no smearing.
Experiment 4: synthesize two primers: 6R and 7F. Use 1 PCR product of experiment 3 as
template. F1-F/6R or 7F/F1R, amplification successful. Combine F1-F/6R and 7F/F1R products, primer F1-F and F1-R, assembly successful.
2) JAD98 gene
Oligo amount 2.5 p mole each in 20 l rxn better than 10 p mole each.
3) Keil-323
Oligo 3’ overlapping 16 bases and 5’ 18 bases.
4) JohnInn 149 and 250
柴油机
起动器Long oligo (111 mer, Alpha DNA), successful.5) K1-537 (Dr. Cavanagh)
Long oligo (115 mer, Alpha DNA).
1 pmole each oligo, touchdown starting at 58C and then 52C, work on 3rd round PCR
Clone screening: clone 3 – 19 base deletion plus other errors (021103B)
黄花菜加工
clone 11 – 5 errors
clone 14 – 18 base deletion plus other 3 errors
clone 20 – 20 base deletion plus other 5 errors
clone R2 – 52 base deletion plus other errors
clone R9 – 76 base deletion plus other 4 errors
clone R21 – 74 base deletion plus other 5 errors
clone R8 – 52 and 20 base deletion plus other errors
clone R22 – 140 base deletion plus other errors
clone R6 – many errors
clone R10 – two nucleotide mutation
Comment: This gene contains 60% GC. There are some short repeated sequences. Increasing annealing temperature may avoid deletion.
6) CCR5 (Dr. Light)
- 1085 bp length, one reaction (12 oligos).
- reverse primer: 80 mer
- 自动变光电焊面罩PCR cloning into pLS vector. Nine clones sequenced. Clone 5 has only one base deletion at 3’ and clone 6 only one base deletion at 5’. - Primers were designed to amplify clone 6/5’ and clone 5/3’ separately. Assembly two fragments in one PCR (primer CCR5-F, CCR5-R, 5’ fragment and 3’ fragment). Clone LSR53 with one base deletion was selected.
- SDM on LSR53
7) 100 (1200 bp, Feb 14, 03)
Oligo 100 mer (Bio S&T),
Experiment 1: 1-14 oligo (2 pmole each), 20 l rxn, 1 or 5 l 1st round PCR product, no amplification; 3非
隔离电源模块
rd round PCR, no amplification. Experiment 2: oligo 1-6, primer 100-F/F1R, successful.
Experiment 3: oligo 7-14, primer F2F/100-R (92 mer), no amplification.
Experiment 4: oligo 7-14 + 100R, primer F2F/100-R
8) HSP169 (456 bp, March 26, 2003)
1 PCR, oligo 1-5; 2 PCR, use oligo 1 & 5 as forward and reverse primers; successful.
9) MBD (243 bp, April 4, 03)
Three oligos (90 –105 mer in length, o/l 16 base), no for and rev primers. Assembly PCR, 6 pmole each oligo.
Digest with NdeI/BamHI, ligate into pET15b vector. Successful.
10) Dr. Drummond (506 bp, May 14, 03)
Use 1st forward primer and last reverse primer for secondary PCR. Successful.
11)
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