Duke University/15 June 2009

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

Latest revision as of 16:39, 21 October 2009

Return

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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