I want to do subcloning of two genes (gene X and gene Y) in pcDNA 3.1+ expression vector. Gene X is having KpnI and NheI restriction site, gene Y having BamHI and NheI restriction sites.
Firstly, I cloned both the genes in 18T-Vector and got positive colony and confirmed by sequencing, then for subcloning I isolated the plasmid and digested it with respective enzymes, but after dual digestion, I am not getting my desired size of my inserts.
Gene X and Y both are approx 1600 bp but after digestion I am getting separate and clear bands at 1000 bp instead of 1600 bp.
Hello Ruheena, did you try with single digestions too? What happened? Did you got just a linear plasmid with the expected mw ?
Did you use the same percentage of agarose gel? and how long you digested your isolated plasmids? Both would affect your results.
Ms. Ruheena: Like suggested by Tiziani, check to see if both of these enzymes are single cutters on your recombinant plasmid. Make sure they don't cut inside the genes!!! Additional sites for either enzymes may be present in the vector backbone, insert, or may be generated at the site of ligation. These are rare events, but may happen, especially if initially not considered . Also, rarely, some enzymes may start showing the star activity.
Unlike PCR cloning by way of poly T ligation, you may try introducing the specific restriction sites directly by designing forward and reverse PCR primers containing the desired sites at the 5' ends, amplify the genes of interest, and then clone directly the restriction enzymes digested PCR products into the expression vectors. Use error proof PCR enzymes,and then sequence verify the recombinants.
Check carefully the restiction maps of both the insert and the plasmid. It is possible you have an internal site cutted by one of the enzymes.
There is for sure an additional cleavage side for one of the enzymes that you did not recognize (control single digest and separation on 1 and 2% gel)
did you check the positive clones in 18T-Vector by restriction digestion? sometimes contaminants (working in the same lab) can give false positive during sequencing.
thanks to all for your interesting answer....but now situation is different
when i checked the gel before gel extraction i was getting bands at 1000bP but when i checked the digested product after gel extraction on same concentration gel, I got both gene (X and Y) bands on expected size (1600bp). Now tell me is this right? and should i have to proceed for ligation?
Make sure to use right size Molecular Markers (DNA ladders). Also, use high quality, 'molecular biology' grade agarose and right buffers (TAE or TBE). To be sure of the genes of interest, perform some appropriate restriction analysis. Finally, you can sequence verify once the recombinants in expression vectors are made.
Thanks for the gr8 suggestion Sir but everything is same like used before (Marker, Agarose and buffer) nothing is change and all are high quality reagents.
i think they are your desired bands. I have met this kind situations for several times in my experiments, and after sequence confirmation, it turned out to be right. And I think the possible reason is that after digestion, your bands were much concentrated, so they looked like a little smaller than expected size, but after gel extraction, it can be treated as your bands were diluted, then they would seemed normal. Hope can help you.
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