Directional cloning - (Feb/20/2007 )
I have done cloning in Teasy vector and checked the insert by DNA isolation and restriction digestion.
The size of my insert is 100bp. Now i want to check for the orientation of the insert by using internal primers but rather than getting one single band with either of the internal primers in combination with T7/SP6 primers i am getting multiple bands, moreover in negative control i always get the DNA band
If anybody can help me to solve this problem
Did you check if your internal primers can also bind on the DNA of the vector? This could also explain the band in your negative control!
better sequence it to confirm orientation.
just sequence it, PCR can sometimes give confusing results in this situation.
yes i will sequence the clone
thanks for the suggestion