Aliquoting Guide for Colony PCR Lab

We have provided sufficient reagents for the number of students you indicated on the Materials Request form, including a bit extra for pipetting errors. All solutions should be aliquoted into the provided 1.5 mL microcentrifuge tubes, unless otherwise indicated.

NOTE: You must mix the primers with the MasterMix no more than three weeks before the PCR laboratory is going to be performed. Thaw the MasterMix and primers, and use as soon as they have thawed. Briefly vortex and flash spin both tubes before mixing. Once MasterMix and primers have been combined, vortex and flash spin before aliquoting. Aliquot on wet ice and immediately refreeze.

You will mix a ratio of 10µL of primers to 12.5uL of MasterMix. You will need to calculate how much to mix using the following formulae:

  1. Total Quantity Needed = 120µL x # lab groups 
  2. Primer Needed = [Total Quantity Needed/22.5µL] x 10µL
  3. MasterMix Needed = [Total Quantity Needed/22.5 µL] x 12.5 µL

For example, if I have 40 lab groups I would do the following:

  1. Total Quantity Needed = 120µL x 40 = 4800µL
  2. Primer Needed = [4800µL/22.5µL] x 10µL = 213.33 x 10µL = 2,133.3µL primer
  3. MasterMix Needed = [4800µL/22.5µL] x 12.5µL = 213.33 x 12.5µL = 2,666.67µL MasterMix

We provide enough extra primer and MasterMix for you to aliquot the amounts shown below.

Part A: Performing PCR

Label tube Contents of tube Aliquot Actually used
PCR MasterMix with Primers (see note above) 120µL 92µL
+ pARA-R plasmid 3µL 2µL
- pARA plasmid 3µL 2µL

Part B: Separate the PCR Products Using Gel ElectrophoresisNOTE: The DNA Ladder used here is 100bp DNA Ladder. This is NOT the same DNA Ladder used in Laboratory 4/4a.

Label tube Contents of tube Aliquot Actually used
M DNA ladder (100bp) 12µL 10µL
Resource category
ABE Lab Specific Resources
Related Lab Foundations - Colony PCR