Engineering BioBrick vectors from BioBrick parts/Colony PCR protocol
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Solutions/reagents:
- PCR SuperMix High Fidelity
- <a name="VF2 primer">VF2 primer
<tab>(5'-TGCCACCTGACGTCTAAGAA-3')</a> - <a name="VR primer">VR primer
<tab>(5'-ATTACCGCCTTTGAGTGAGC-3')</a> - <a name="colony suspension">colony suspension
<tab>(1 colony diluted in 100 ?l water)</a> - de-ionized water
Parameters:
- X - concentration of primers (µmole/µl)
Equipment:
- Thermocycler
- Sterile 0.6-ml tubes
Steps:
- PCR mix
Use the following table as a checklist for preparing the reaction in sterile 0.6-ml microcentrifuge tube (1):
<thead></thead><tbody></body>PCR SuperMix High Fidelity VF2 primer VR primer colony suspension de-ionized water Colony PCR 9 µl 0.125/X 0.125/X 1 µl Make up the volume to 20 µl - PCR conditions
Program a standard thermocycler to run the reaction using the following parameters:
Initial denaturation- Denature: 95°C, 15 mins
- No. of cycles: 39
- Denature: 94°C, 30 secs
- Anneal: 62°C, 30 secs
- Elongate: 68°C, 3.5 mins
- Elongate: 68°C, 20 mins
- Hold: 4°C, until removed from machine
TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 3 hrs, 35 mins
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