Engineering BioBrick vectors from BioBrick parts/Colony PCR protocol

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Solutions/reagents:

• PCR SuperMix High Fidelity
• VF2 primer
  
  (5'-TGCCACCTGACGTCTAAGAA-3')
• VR primer
  
  (5'-ATTACCGCCTTTGAGTGAGC-3')
• colony suspension
  
  (1 colony diluted in 100 µl water)
• de-ionized water

Parameters:

• X - concentration of primers (µmole/µl)

Equipment:

• Thermocycler
• Sterile 0.6-ml tubes

Steps:

1. PCR mix
   Use the following table as a checklist for preparing the reaction:
   
   

   	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

2. PCR conditions
   Program a standard thermocycler to run the reaction using the following parameters:
   Initial denaturation
   
   • Denature: 95°C, 15 mins
   Thermocycling
   
   • No. of cycles: 39
   • Denature: 94°C, 30 secs
   • Anneal: 62°C, 30 secs
   • Elongate: 68°C, 3.5 mins
   Termination
   • Elongate: 68°C, 20 mins
   • Hold: 4°C, until removed from machine