View source for EcoRI digest of plasmid

Since the TOPO pcr2.1 plasmid has EcoRI cut sites on either side of the PCR insert, you can perform a restriction digest with EcoRI enzyme to check the size of the insert after plasmid extraction. This ensures that the plasmid you extracted from the E. coli liquid culture is consistent with your colony PCR results. (Remember that your culture could become contaminated with something else between colony PCR and plasmid extraction, so this digest serves as a verification before electroporation. 


1. Set up a 10uL reaction in a PCR tube for each sample that includes:
<br>- DNA  150ng (check the concentration of your plasmids to determine the required volume)
<br>- 1 uL 10X rCutsmart Buffer
<br>- 0.5 uL EcoRI-HF 
<br>- PCR water to 10uL

2. Incubate for 30 min at 37C (10-15 is usually enough to digest 150ng DNA if you are short on time). Note that other enzymes will require different buffer, incubation temp, and/or incubation time. For partial digest, run no more than 30sec before moving to step 3, and ensure that a heat block or water bath is prepared at 65C for rapid inactivation. 

3. Heat inactivate the restriction enzyme at 65C for 20 min. 

4. Mix with 6X purple loading dye and run on a 0.8% agarose gel along with appropriate DNA ladder to determine the size of the insert.