FAQ: What problems can be encountered in the restriction digest that can cause ligation using T4 DNA Ligase or subsequent transformation to fail?

* The restriction enzyme did not cleave efficiently. If cleaving near the end of a PCR * fragment leave at least 6 bases past the restriction site. Test the restriction enzyme on a control substrate.
* The restriction enzyme was not completely inactivated. Phenol/ETOH purify the DNA if the enzyme cannot be heat inactivated.
* Star activity from the restriction digest cleaved the vector or insert. Check the DNA on a gel. If there is an extra band, reduce the amount of enzyme or time for the restriction digest.
* The DNA or restriction enzyme contained exonuclease or phosphatase that damaged the ends. Phenol/ETOH purify the DNA. Check the enzyme QC data and notes. If the ligation QC is poor or the exonuclease level is high reduce the amount of enzyme or incubation time.
* The PCR process is covered by patents owned by Roche Molecular Systems, Inc.