Engineering BioBrick vectors from BioBrick parts/Colony PCR
Materials
PCR SuperMix High Fidelity
VF2 primer (
5''-TGCCACCTGACGTCTAAGAA-3'')VR primer (
5''-ATTACCGCCTTTGAGTGAGC-3'')Deionized, sterile H2O
strip of PCR tubes
2-log DNA ladder
0.8% E-Gel® from Invitrogen Corporation in Carlsbad, CA
Equipment
DNA Engine OPTICON\texttrademark from MJ Research, Inc. (now Bio-Rad Laboratories, Inc., Hercules, CA)
E-Gel® PowerBase™ v.4 from Invitrogen Corporation in Carlsbad, CA
Alpha Innotech FluoChem™ 880 gel imaging system
Procedure
PCR mix
9 μL PCR SuperMix High Fidelity
6.25 pmoles VF2 primer
6.25 pmoles VR primer
1 μL colony suspension
dilute 1 colony in 100 μL water
PCR conditions
95°C for 15 minutes
94°C for 30 seconds
62°C for 30 seconds
68°C for 3.5 minutes
Repeat 2-4 39 times.
68°C for 20 mins
4°C forever
Agarose gel electrophoresis
Dilute the reactions four-fold with water.
Perform an agarose gel electrophoresis of 20 μL of each diluted reaction using a 0.8% E-Gel®.
Also electrophorese 1 μg of 2-log DNA ladder to verify the length of each PCR product.
Image gel with 302 nm transilluminating ultraviolet light using an ethidium bromide camera filter and an exposure time of 614 milliseconds.
BioCoder version
Following is the Engineering BioBrick vectors from BioBrick parts/Colony PCR protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi
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综述
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