Protocol: Template Preparation 

1. First, one must have a monoclonal E Coli colony source. This may be one of the following three things:
   1. A glycerol stock made from a SINGLE colony on an agar plate
   2. An overnight culture in LB media (+ antibiotics if appropriate) started from a SINGLE colony on an agar plate
   3. A SINGLE colony on an agar plate
2. Label a sterile eppendorf with the strain number and assign a unique colony number for each colony picked. Pipet 20 uL of sterile water into the tube. If the e coli is coming from sources (1) or (3), use a clean, sterile pipet tip to scrape a tiny bit of bacteria (even the very smallest amount is okay) and then pipet up and down into the waiting 20 uL. If the e coli is coming from source (2), pipet 1 uL of the LB culture into the water. Mix well.
3. This water/colony mixture will be the “template” for your PCR reaction. Next, set up your reaction mixture and run the cycler protocol. Follow this up by running a gel to examine the PCR amplicon lengths.

Reaction Preparation

Reaction Mixture (20 uL):

1. 1 uL Template
2. 10 uL 2x Taq Master Mix (Long term storage in freezer, will last one month at 4C)
3. 1 uL FWD Primer (@ 10 uM)
4. 1 uL REV Primer (@ 10 uM)
5. 7 uL H2O

Cycler Protocol

The cycler protocol is fixed except for the extension time. Set the extension time for 1 min per kb of desired amplicon at 68C, plus a little bit of buffer time. For example, for a 2 kb amplicon, set the extension time to 2 min 30 seconds.

1. 95ºC for 6 minutes
2. 30x [95ºC for 30 sec, 55ºC for 30 sec, 68ºC for 1 min/kb amplicon]
3. 68ºC for 20:00 min
4. Hold at 4ºC