Engineering BioBrick vectors from BioBrick parts/Colony PCR
From OpenWetWare
Jump to navigationJump to search
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
Text Output
Engineering BioBrick vectors from BioBrick parts/Colony PCR protocol
Source Code
Engineering BioBrick vectors from BioBrick parts/Colony PCR protocol - source code