Colony PCR

Choose and mark 28 well-separated colonies on your plates (preferably from only the 50 uL plate but can use both if needed). Circle each colony and label with numbers 1-28. 

Recipe - (mix all together in order given, aliquot into PCR tubes in 20 uL each)

Reagent	Per 20 uL reaction	For all 28 reactions:

For 9.5 uL water in each 20 uL reaction, add 266 uL (***Add more, - 275-285 uL to account for pipetting error)

Primer 1 **100 uM	0.25 uL	7 uL

Primer 2 ** 100 uM	0.25 uL	7 uL

2x Green MasterMix	10 uL	280 uL (found in the -30)

'''

To each 20 uL aliquot:

 Gently touch the tip of a pipet (pipet depressed to ~ 5 uL) to the requisite colony to get some cells on pipet tip. Carefully place pipet into PCR tube and pipet up and down 6-7 times to suspend cells in solution. Spin down.

 

PCR program:

  '''

** Don’t forget to record the volume of the reaction in the PCR program BEFORE running.

When PCR run is complete, run your samples using Gel Electrophoresis and look for bands of the estimated size. If a colony, or a few colonies, on your gel show the correct predicted length, this means theses colonies were successfully transformed.

The next steps for your colonies will be:

Growing Bacterial Colonies in LB

Sending out DNA for Sequencing