I was going through your problem, and reverse amplification chances are about 50% in TA cloning technique....and i came across an article "One-step DNA Fragment Assembly and Circularization for Gene Cloning" (http://www.horizonpress.com/cimb/abstracts/v12/11.html). Hope it helps :)

Design a pair of PCR primers, with one annealling to the vector and one on the insert, in such a way that they would only make the product if the desired orientation is present. You can also design a complementary pair as a control to look at the other orientation. This way, you can perform colony PCR to identify the orientation of your insert straight out of the plate.

I generally grow up 6 colonies, miniprep, and cut with 2 unique restriction enzymes.

Find a restriction site near the 5' or 3' end that is unique to your insert and use one of the unique sites in the vector to look for the correct sized fragment. The orientation of the cut site will not effect the restriction cleavage as they are palindromic.

e.g. vector Xho1 -----5' -----'hindIII---------------3'---vector correct orientation would give a fragment of 10 dashes and remaining vector.

vector Xho1 -----5'---------------'hindIII3'-----3'---vector incorrect orientation would give a fragment of 20 dashes and remaining vector.

Thanks all.

I generally run PCR with expression primers with 1 microlitre of sub-culture, to confirm the plasmid with insert. My forward expression primer has BamHI site and reverse primer has HindIII site. PCR with the expression primers is normal. But once i sequence to confirm the insert and any mismatches, i sometimes find the insert in reverse orientation i.e.

5' -M13 Reverse primer (TA vector)-------HindIII-insert--------BamHI---3'. Whether such an orientation (reverse) is feasible for correct protein expression.

So i assume that you're tryimg to use the restriction sites on the topo vector to continue your cloning with. What TA cloning vector did you use as backbone? Invitrogen's pCR2.1? pCR4? Shouldn't be 2.1 since the BamHI and HindIII site are on the side of TA insert site.

Another way to totally avoid this screen is just to start amplifying you goi with pcr primers with desired restriction sites incorporated into each f and r primers in the right position. That way you wouldn't have to worry of orientation after TA cloning. Just make the plasmid and digest.

Thanks Dr.

True, iam using Invitrogen's pCR2.1 TOPO TA Cloning vector having BamHI and HindIII sites. I usually send the purified plasmid for sequencing. Do you suggest me to amplify the goi with pcr primers designed with BamHI and HindIII restriction sites towards screening the correct orientation of the insert. Definitely, i have tried digesting with BamHI and HindIII but it seems HindIII is not cutting and therefore my insert is not of expected size after double digestion, but a little higher up. Lately we have designed expression primers with EcoRI restriction site and hope it works. Any suggestions are welcome on this topic.