Team:Hawaii/PCR Amplification of pRL1383a

From 2008.igem.org

Revision as of 23:47, 3 July 2008 by MargaretRuzicka (Talk | contribs)
Projects Events Resources
Sponsors Experiments Milestones Protocols
Notebook (t) Meetings (t)

Contents

PCR Amplification of pRL1383a

  • Several regions of pRL1383a will be amplified with BioBrick based primers. These components will be used later in the construction of a pRL1383a BioBrick based vector. These parts include the aadA region of the omega interposon, the origin of vegetative replication (oriV), the mobilization proteins, and the replication proteins.

Methods

Materials

  • Taq polymerase: Accusure(hot start, 68°C) and Red Taq
  • Nanopure water
  • forward/reverse primers for each BioBrick
  • pRL1383a as template
PCR Running Conditions
Duration of Time T° 5 cycles T° 30 cycles Purpose
10 minutes 95°C heat activate taq
30 seconds 95°C 95°C denaturation
30 seconds 48.1°C, 53.4°C, 57.9°C 59°C, 60.6°C, 61.9°C annealing
5 minutes 68°C 68°C extension (1.5 minutes per kb, go with longest)
10 minutes 68°C 68°C finishing the extension
infinity 4°C 4°C (until you remove product)

Results


Discussion

  • What was learned and how to do future experiments differently.
Insanity is doing the same thing over and over again and expecting different results. - Albert Einstein


[http://manoa.hawaii.edu/ Sponsor_UHM.gif][http://manoa.hawaii.edu/ovcrge/ Sponsor_OVCRGE.gif][http://www.ctahr.hawaii.edu Sponsor_CTAHR.gif]