Smolke:Protocols/Screening

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).

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