Smolke:Protocols/Screening

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Josh's Protocol Sample Preparation 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 5 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 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 parental 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.