What is the suitable expression vector for expressing 2500bp insert in bacterial system?

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jesuis vert
jesuis vert's picture
What is the suitable expression vector for expressing 2500bp insert in bacterial system?

hi everyone,I am working on a gene size 2500 bp. I have successfully cloned it in pgemt vector and now i want to express it in bl21 cells. But on ligating this insert with pet28a vector, I am getting no colonies. I have tried ligation reaction replacing buffer and enzyme (NEB t4 DNA ligase) with new aliquots, but results are still same. I wonder if problem arising due to vector size pet28a which is of only 5400 bp, and insert size is 2500bp. So shall I use larger size vector (eg. pet22b) to express larger size gene. Whether large size vector is required for large size insert. If anyone has faced same problems, kindly suggest me how to deal with it.Thanking you

Donald Alcendor
Donald  Alcendor's picture
What is the suitable expression vector for expressing 2500bp ins

 Reaction mixes does not seem to be the problem.  The vector size is also not an issue.  Contaminants associated with the vector DNA and the insert fragment need to be removed.Make sure restriction enzymes are working and you're getting the right size bands by agarose gel electrophoresis.1. I would suggest that you run both the vector and the insert on an agarose gel, cut out the fragments, gene clean the both fragments in 20 ul of Tris EDTA pH 7.5.  Use a vector/insert ratio 1:3 and use 5ul of the ligation mixture to trasfect E.coli bl21 competent cells. Plate the cells and look for colonies.  Incubate the plates for 24-48 hours.  If this gene product is toxic to the bacteria this could also be a problem.The pet directional vectors should be fine for this cloning procedure.  Make sure the competent bl21 cells are good and haven't thawed.  They should be at -80C at all time until used.

jesuis vert
jesuis vert's picture
What is the suitable expression vector for expressing 2500bp ins

thank you for your reply Sir Alcendor.I have gel eluted both gene & vector using promega kit and got O.D.= 27.8 ng/ul & 10.7 ng/ul respectively. I have used ratio both 1:3 & 1:5 but dint get results. I have also checked vector & gene running on 1% agarose gel then gel eluted it. if I suppose, for instance, my gene is toxic then, what preventive measures I should consider?I have a doubt that O.D. of vector is very less. Do you want to comment on this?