problem with gateway cloning: missing part of insert - (Sep/13/2013 )

Currently I am trying to clone an attB PCR product into gateway pDONR221. After performing the BP reaction I got some few clones which I checked by M13 PCR and they seemed to be positive. However, sequencing revealed that quite a substantial part of the gene is missing, i.e. starting with a PCR product of almost 600 bp, I end up with only a bit more than 300 bp inserted into the vector.

Subsequently I cloned the attB PCR product into pGEM, checked it by sequencing and got exactly what I expected: my 600 bp sequence with the attB sites on both ends. So I think it is a problem with the BP clonase reaction?

Has anyone encountered similar problems? Any ideas?

BLAST your attB sites against your insert once

I checked this already, but I don't find anything similar to the attB sites within the insert

taphrina on Fri Sep 13 09:58:26 2013 said:

Currently I am trying to clone an attB PCR product into gateway pDONR221. After performing the BP reaction I got some few clones which I checked by M13 PCR and they seemed to be positive. However, sequencing revealed that quite a substantial part of the gene is missing, i.e. starting with a PCR product of almost 600 bp, I end up with only a bit more than 300 bp inserted into the vector.

Subsequently I cloned the attB PCR product into pGEM, checked it by sequencing and got exactly what I expected: my 600 bp sequence with the attB sites on both ends. So I think it is a problem with the BP clonase reaction?

Has anyone encountered similar problems? Any ideas?

Do you find the reason and solution? I am having the same problem. Please contact me. Thanks.