Knight:Colony PCR

See Colony PCR for general information about this protocol and other variants

Materials

 * Sterile 0.6mL plastic tubes
 * LB-agar plate with appropriate antibiotic
 * Oligonucleotide pair (usually VF2 and VR)
 * PCR supermix
 * PCR machine
 * Pipetman
 * Sterile pipet tips

Picking colonies

 * 1) Prepare one sterile 0.6mL tube with 20&mu;L ddH2O for each colony you intend to pick.
 * 2) Prepare LB-agar plate with appropriate antibiotic to use as index plate.
 * 3) Pick single colony using a pipetman with sterile tip. The pipettor should be set to 3&mu;L
 * 4) Inoculate tip with colony into tube. Pipet up and down to ensure cells are transferred to tube.  Pipet 3&mu;L of cells suspended in water onto index plate.
 * 5) *Alternatively, you can use a multi-channel pipettor to make an index plate once you are done picking colonies.
 * 6) Repeat for as many colonies as you intend to pick.
 * 7) *For routine assemblies, I pick 4-8 colonies per construct.

Reaction mixture
1X Reaction
 * 9 &mu;L PCR supermix
 * 0.25 &mu;L 40&mu;M VF2
 * 0.25 &mu;L 40&mu;M VR
 * 0.5 &mu;L colony template

PCR conditions

 * 1) 95&deg;C for 15 mins
 * 2) 94&deg;C for 30 secs
 * 3) 56&deg;C for 30 secs
 * 4) 68&deg;C for 1 min per kb of expected product
 * 5) *I typically round up for this step. i.e. For a 3.6kb construct, I used a 4 min elongation time.  It seems to help to be a bit generous with the elongation time.
 * 6) Repeat 2-4 39 times.
 * 7) 68&deg;C for 20 mins
 * 8) 4&deg;C forever

Run a gel to determine amplification product length.

BioCoder version
Following is the Knight:Colony PCR protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi

Text Output
Knight:Colony PCR protocol

Source Code
Knight:Colony PCR protocol - source code