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The true test of a first-rate mind is the ability to hold
two contradictory ideas at the same time.
F. Scott Fitzgerald
Day 5: DNA design
Assignments Due
Overview of Experiment
In today's lab, you use PCR to amplify plasmid DNA and mutate
the ribosomal binding site for GFP.
A high fidelity DNA polymerase will be used to ensure that
the DNA sequence is correct after amplification. You
will use Phusion® Hot
Start Flex 2X Master Mix (New England Biolabs
(NEB), Ipswich, MA).
A. Reaction setup: in a 0.5 ml tube, add
the reaction components in the following order for a 50 µl
reaction
- 20.5 µl nuclease-free (NF) water
- 1 µl forward (F) primer (0.5 µM stock)
- 1 µl reverse (R) primer (0.5 µM stock)
- 1.5 µl DMSO
- 1 µl I13522 plasmid DNA (template DNA)
- 25 µl Phusion Hot Start Flex 2X Master Mix
*Also set up a negative control reaction using 1 µl water
B. Cycling conditions:
- Initial Denaturation:
98°C for 30 sec
- 30 cycles:
98°C for 10 sec (denaturation)
62°C for 30 sec (annealing)
72°C for 2 min (extension)
- Final Extension:
72°C for 10 minutes to complete the run
- HOLD at 4°C indefinitely
Copyright, Acknowledgements,
and Intended Use
Created by B. Beason (bbeason@rice.edu),
Rice University, 21 November 2007
Updated 6 November 2013