Posted 01 March 2011 - 08:38 AM
Does anyone know what might be causing this decrease?
Posted 01 March 2011 - 08:29 PM
Different amount of vector in the ligation mix, results in different number of colonies.
Was the PCR fragment purified before being added to the ligation mix?
PCR buffer does contain detergent which does kill bacteria cell if carried over into the ligation mix.
Was the vector gel purified?
1hr in SAP is rather long. If the vector is not gel purified uncut DNA can get into the ligation mix. Denature plasmid DNA is resistant to digest but will transform just fine.
Posted 07 March 2011 - 12:41 AM
If you didn't notice that, the best way is clone you gene to TA vector first, so you can do the sequencing very fast, then select the correct one and use the normal double digestion to ligate your gene to the desired vector.
Edited by Quasimondo, 07 March 2011 - 12:41 AM.
Posted 14 March 2011 - 01:42 AM