Ligation of digested plasmid to hairpin oligo
Posted 10 September 2009 - 08:35 AM
Posted 10 September 2009 - 03:55 PM
Posted 14 September 2009 - 05:37 AM
Posted 14 September 2009 - 09:06 PM
Posted 15 September 2009 - 05:26 AM
Posted 16 September 2009 - 05:34 AM
You don't say how you set up your ligation, but a very easy trick to try is: set up everything EXCEPT the ligase and warm up the mixture (with such a small overhang you wouldn't need to warm it up much) and let it cool slowly when it hits around room temp (or cooler if you like) add your ligase. The idea being, perhaps before ligation (esp. if you set it up on ice) your BS plasmid is already based paired in a concatamer and when you add your ligase your oligo never had a chance to bind. Easy enough to try anyway. Warren..