Aliquot 50 uL sterile water into sterile PCR tubes, one tube per colony.
Using a P20, scape off a colony and resuspend it in the water.
Use 1 uL of this cell suspension as your PCR template
Tubes can be stored at 4C for several days. Use 5 uL of cell suspension to inoculate a 5 mL overnight culture.
Reaction Mixture
Per colony:
1 uL cell suspension
0.2 uL dNTPs
0.2 uL FWD and REV primers
1 uL 10x Taq Buffer (with MgCl2)
0.2 uL 1:10 Taq
7.2 uL water
Note: Make a master mix of everything except the template. Aliquot 9 uL into a separate set of PCR tubes. Then transfer 1 uL of cell suspension to each tube. You can use a multichannel pipet to transfer up to eight templates at once.
Run PCR as normal, with the first step changed to a 7 minute incubation at 95C (5 minutes to lyse + 2 minutes to denature DNA).
Yvonne's Protocol
Procedure
Warm up necessary number of LB (+ appropriate antibiotics) plates for restreak
Make master mix; include 1 or 2 extra volumes to account for errors from repetitive pipetting. Remember to account of positive and negative control samples. For each sample:
19.5 ul water
2.5 ul 10x Taq buffer
1.25 ul 50 mM MgCl2
0.5 ul Fwd primer
0.5 ul Rev primer
0.5 ul dNTP
Line up necessary number of PCR tubes
Take out pre-warmed LB plate and label with colony numbers
Add 0.25 ul 1:10 Taq DNA polymerase for each sample to master mix; pipette or invert tube to ensure thorough mixing
Aliqout 25 ul of master mix to each tube
Pick up each colony with a toothpick, dip into PCR tube (containing master mix) and swirl, then restreak on LB plate
Run PCR as normal, except the first step is changed to 95C for 5 min for cell lysis
Note: The second step is 94C for 2 min, and this step is repeated each cycle while the first lysis step is not
Put restreaked plate back in incubator; restreaked colonies should be ready for inoculating liquid cultures in 4 hours
Load 15 ul of each PCR product on agarose gel for analysis
Notes
Ideally, use primer sets that would generate a band for both positive and negative colonies but with different sizes.
Should always include a negative control using the parent vector, no DNA template, or another appropriate choice. This would let you know if you have primer dimer or other nonspecific amplification.
Should include a positive control if possible. This is especially important if your chosen primers would only generate a band for positive but not negative colonies.
I use toothpicks whenever possible. They are much cheaper and more environmentally friendly (I believe) than pipet tips.
