Posted 20 June 2011 - 05:06 PM
Posted 20 June 2011 - 10:27 PM
You can do also restriction enzyme digestion.
Posted 21 June 2011 - 12:12 AM
Posted 21 June 2011 - 02:26 AM
Seems like you have problem with ligation..even you have good colonies number but that all without insert..(mean there's nothing wrong with your transformation)
maybe the pcr product has no restriction site at the end of the fragment...? some people designing a fragment with few base pair (4-5bp) before RE site in their primers.. so if you did same thing make sure u digest it first...
and if u did that and no problem with digestion, maybe the ligation mixture is not good... for ligation...how long do u incubate the ligation mixture?at what temperature...try to extend it longer at 16C...Wish of luck