Engineering BioBrick vectors from BioBrick parts/Colony PCR: Difference between revisions
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*strip of PCR tubes | *strip of PCR tubes | ||
*[http://www.neb.com/nebecomm/products/productN3200.asp 2-log DNA ladder] | *[http://www.neb.com/nebecomm/products/productN3200.asp 2-log DNA ladder] | ||
*0.8% E-Gel® | *0.8% E-Gel® from Invitrogen Corporation in Carlsbad, CA | ||
==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) | ||
*E-Gel® PowerBase™ v.4 from Invitrogen Corporation in Carlsbad, CA | *E-Gel® PowerBase™ v.4 from Invitrogen Corporation in Carlsbad, CA | ||
*Alpha Innotech FluoChem™ 880 gel imaging system | *Alpha Innotech FluoChem™ 880 gel imaging system | ||
Revision as of 09:00, 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® 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.