Engineering BioBrick vectors from BioBrick parts/Colony PCR: Difference between revisions
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#Also electrophorese 1 μg of 2-log DNA ladder to verify the length of each PCR product. | #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. | #Image gel with 302 nm transilluminating ultraviolet light using an ethidium bromide camera filter and an exposure time of 614 milliseconds. | ||
==BioStream version== | |||
Following is theEngineering BioBrick vectors from BioBrick parts/Colony PCR protocol in BioStream, 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 BioStream (see Source code). More information about BioStream can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth|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]] | |||
[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category:Escherichia coli]] | [[Category:Escherichia coli]] | ||
[[Category:DNA]] | [[Category:DNA]] |
Revision as of 23:23, 30 September 2009
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.
BioStream version
Following is theEngineering BioBrick vectors from BioBrick parts/Colony PCR protocol in BioStream, 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 BioStream (see Source code). More information about BioStream 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