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 

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

Reaction Mixture Preparation

1. 5 uL 10X Digest Buffer
2. 1 uL of each digestion enzyme up to maximum of 2.5 uL
3. 2000 ng of desired cut fragment up to max volume of 50 - enzyme volume (cutsmart+digestion enzyme volumes)
4. Fill with milliQ water to 50 ul

Digestion Protocol 

1. Incubate the reaction at 37ºC for 1 hour.
2. The digest buffer is specific to the enzyme and it stated on the enzyme product description - look it up on NEB’s webpage for each enzyme. It is generally “Cutsmart.” For digests with multiple enzymes, use NEB’s tool to identify the right buffer.
3. Enzymes are stored in glycerol, but glycerol can inhibit digestion reactions. To keep the volume to 2.5 uL, use 1 uL of 2 enzymes. For three enzymes, consider using just 0.5 uL of the most active one or reducing the volume of all three to 0.8 uL.
4. You want there to be at least 2000 ng of the fragment you want to isolate in the end. This is because DNA digestion products must be purified, a process that is only about 50% efficient. Isolating 2000 ng ensures you have enough DNA in the end for cloning. To calculate the volume, use the DNA concentration and the relative fraction of the plasmid that is the fragment you want. For example: for a 10 kb plasmid at 1000 ng/uL with a 1kb fragment I want to cut out, I’d want to do the following calculation: