Help!!! Nonspecific insert in TOPO TA cloning - is it possible? (Jul/13/2005 )
I tried to clone part of human gene using TOPO TA kit from invitrogen. First desired band of PCR product from gel was extracted and purified. Then it was cloned using TOPO TA kit. Screening of colonies revealed bands of expected size. But plasmid sequencing showed that between gene-specific primers there is a sequence which doesn't have common with cloned gene. Can You help and suggest where is the problem, what can be the reasons and how to solve it. Thank You beforehand
I have got the same problem with TOPO sequencing kit. I cloned my PCR product into the vector, transfected cells, got very low number of colonies, which had the right size insert (amplified with primers specific for vector as well as for the PCR product). However, when my plasmids were sequenced I got completely different sequence. BLAST search gives sequence of fragment of different plasmids. Sequencing of the PCR product gave correct sequence. Does anyone have any idea what is going on? Any suggestions will be appreciated. Thanks.
Given that you are seeing your gene specific primers and the middle sequence is non-specific I would suggest that the problem lies in your PCR it self. Can you sequence your PCR product or at least restriction map the PCR product. Is there any chance of redesigning your PCR primers and check there specificity by BLASTing.
Usually when you BLAST and get a varied array of different plasmids it suggests that your plasmid doesn't have insert, easy to confirm with a restriction map. This means two things first: your ligation probably hasn't worked and needs to be optimised and second: your colony PCR screen may be contaminated did you get negative colonies in your screen as well??
Thank You for Your reply. Analysis of primers against Blast showed that they were specific for chosen gene. Moreover they worked normal in RT-PCR and in nested RT-PCR in rather strict conditions (annealing at 65). And unfortunately, RQ-PCR analysis of plasmid with primers/probes localised between our primers gave negative results, although again this set of primers/probe also has good specificity and CV