Duke University/15 June 2009

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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