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June 15, 2009
- E: PCRs with Bioplastics #1 (fragments 1 and 2) and #3 (fragments 3 and 4) as templates, doubled volume, eliminated annealing step, varied extension (plus annealing) time based on fragment size, Gels to check PCRs
5x HF buffer 10 ul
dNTPs 4 ul
forward primer 2.5 ul
reverse primer 2.5 ul
DNA template (plasmid) 1 ul
Phusion DNA polymerase 0.8 ul
H2O 29.2 ul
50 ul
98°C 30s 1x
98°C 15s
72°C varies 35x
72°C 5m
4°C infinity 1x
Annealing Time: 30s
Extension Time: 15 s * size/length (kb)
Fragment # Name Size/Length (bp) Time (s)
1 pASKphaA 4215 100
2 phaB 896 50
3 Termcat2 1701 60
4 phaC 1872 65
- R: Bands for fragments 1 and 2, No bands for fragments 3 and 4
- C: Cut out bands of fragments 1 and 2, PCR again to obtain fragments 3 and 4
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