cloning PCR fragment
Posted 01 September 2004 - 01:09 AM
Please help me
Posted 01 September 2004 - 09:25 AM
Posted 01 September 2004 - 01:45 PM
To check the restriction efficiency, use T4 kinase to label the 5' ends with 32P. Then cut one aliquot with Sal I, one aliquot with BamH I, and a third aliquot with both enzymes. Run the products on a gel with uncut labeled DNA and quantify the amount of label remaining on the insert.
Option 1: get a TA cloning kit and forget about reestriction cuts.
Option 2: polish the ends and then cut with BamH I. Ligate into vector cut with BamH I and a blunt-end restriction enyme.