Engineering BioBrick vectors from BioBrick parts/Colony PCR: Difference between revisions
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(New page: ==Materials== *[http://www.invitrogen.com/content/sfs/manuals/11306016.pdf PCR SuperMix High Fidelity] *VF2 primer (<code>5'-TGCCACCTGACGTCTAAGAA-3'</code>) *VR primer (<code>5'-ATTACCGCC...) |
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| Line 6: | Line 6: | ||
*Deionized, sterile H<sub>2</sub>O | *Deionized, sterile H<sub>2</sub>O | ||
*strip of PCR tubes | *strip of PCR tubes | ||
*[http://www.neb.com/nebecomm/products/productN3200.asp 2-log DNA ladder] | |||
*0.8% E-Gel® | |||
==Equipment== | ==Equipment== | ||
*DNA Engine OPTICON\texttrademark from MJ Research, Inc. (now Bio-Rad Laboratories, Inc., Hercules, CA). | *DNA Engine OPTICON\texttrademark from MJ Research, Inc. (now Bio-Rad Laboratories, Inc., Hercules, CA). | ||
*Alpha Innotech FluoChem™ 880 gel imaging system | |||
==Procedure== | ==Procedure== | ||
| Line 30: | Line 33: | ||
#68°C for 20 mins | #68°C for 20 mins | ||
#4°C forever | #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. | |||
[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category:Escherichia coli]] | [[Category:Escherichia coli]] | ||
[[Category:DNA]] | [[Category:DNA]] | ||
Revision as of 08:57, 18 March 2008
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®
Equipment
- DNA Engine OPTICON\texttrademark from MJ Research, Inc. (now Bio-Rad Laboratories, Inc., Hercules, 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.
