PCR targeting system in Streptomyces coelicolor ... - Streptomyces UK

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Fig.2: Designing PCR primers for making an 39 nt from sense strand ending in ATG or GTG start codon If sequence with >30 matches occurs in cosmid clone, move → 3n nt to maintain frame NNNN .... ATG NNNN .... TAC 59 nt upstream primer NNNN .... ATG 20nt * (the example illustrates a complete deletion) Gene to be deleted FRT Apra R ± oriT FRT Cassette FLP recombinase (BT340) 59 nt upstream primer NNNN .... ATG 20nt TGA …. NNNN NNNN .... TAC 19nt ACT …. NNNN 81 bp scar (20bp + 19bp priming sequence + 42bp FLP core recombination site (see Fig.3); no in frame STOP) 6 in-frame deletion 39 nt from anti-sense strand ending in Stop codon ← move –3n nt if necessary 58 nt downstream primer TGA …. NNNN ACT …. NNNN * 20 and 19 nt sequences are identical in all cassettes • 20 nt: 5` ATT CCG GGG ATC CGT CGA CC 3` • 19 nt: 5` TGT AGG CTG GAG CTG CTT C 3` 19nt * ACT …. NNNN 58 nt downstream primer
3 PCR amplification of the extended resistance cassette All PCR amplifications are performed using the Expand high fidelity PCR system according to the manufacturer’s instructions (Roche). Reaction conditions: • Primers (100 pmoles/μl) 0.5 µl each 50 pmoles each • Template DNA (100 ng/μl) 0.5 µl 50 ng ≈ 0.06 pmoles • Buffer (10x) 5 µl 1 x • dNTPs (10 mM) 1 µl each 50 µM each • DMSO (100 %) 2.5 µl 5% • DNA polymerase (2.5 U/μl) 1 µl 2.5 Units • Water 36 µl • Total volume 50 µl Cycle conditions: 1. Denaturation: 94°C, 2 min 2. Denaturation: 94°C, 45 sec 3. Primer annealing: 50°C, 45 sec 10 cycles 4. Extension: 72°C, 90 sec 5. Denaturation: 94°C, 45 sec 6. Primer annealing: 55°C, 45 sec 15 cycles 7. Extension: 72°C, 90 sec 8. Final extension: 72°C, 5 min 5 µl of the PCR product is used for analysis by gel electrophoresis. The expected sizes are 78 bp larger than the sizes of the disruption cassettes listed in Table 1 (because of the 2 x 39 bp 5´-primer extensions). The remaining 45 µl of the PCR product is purified using the Qiagen PCR purification kit according to the manufacturer’s instructions. The PCR product is finally eluted from the columns with 12 µl of water (~200 ng/µl). 7
Page 1 and 2: PCR targeting system in Streptomyce
Page 3 and 4: Fig. 1: Flowchart of gene disruptio
Page 5: 2 Design of long PCR primers For ea
Page 9 and 10: 5 PCR targeting of the S. coelicolo
Page 11 and 12: The gene disruption is confirmed by
Page 13 and 14: 6 Transfer of the mutant cosmids in
Page 15 and 16: Antibiotic • Typically, ~ 10 % of
Page 17 and 18: 1. Grow E. coli DH5α/BT340 overnig
Page 19 and 20: quickly while unicellular contamina
Page 21 and 22: The λ RED recombination plasmid pI
Page 23 and 24: LOCUS pIJ773 4334 bp DNA CIRCULAR S
Page 25 and 26: amp pIJ778 4377 bps priming site pr
Page 27 and 28: 2461 TCACTGACTC GCTGCGCTCG GTCGTTCG
Page 29 and 30: LOCUS pIJ780 4449 bp DNA CIRCULAR S
Page 31 and 32: Strains: Strain designation: BW2511
Page 33 and 34: Agar Media Note: MS medium (Mannito
Page 35 and 36: Mannitol Soya flour Medium (MS) Hob
Page 37 and 38: 2 X YT medium Difco Bacto tryptone
Page 39: The CD-ROM includes: Literature 1.

PCR targeting system in Streptomyces coelicolor ... - Streptomyces UK

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