I'm transforming CaCl2 competent 50 ul E. coli DH5alpha with 1 ul of pET28a and pET30 at 42 oC for 45 seconds. I added 1 ml LB and incubate at 37 oC with shaking for 1 hour and then plated on LB agar having 50 ug/ml Kanamycin. The growth is very slow with pin pointed colonies. While same competent cells transformed with pET15b shows good growth. What could be the possible reason.
Dear Kumar,
Please check the type of antibiotic resistance genes in the following vectors pET28a and pET30. Maybe, somehow they are not Kanamycin resistant.
Good Luck,
Reduce the kanamycin concentration around 35 ug/ml. What is the concentration of plasmid you are using for transformation. You may need to increase it to 2-3 ul if it is very less concentrated.
In my experience, kanamycin resistance-based plasmids (like your pET28 and 30) generally cause a slower bacterial growth than ampicilin resistance based plasmids (like your pET15). This would be mostly due to the lag phase being longer (or actually, just present to begin with).
- Ampicillin basically kills cells when they divide, thus cells have some time to express the resistance protein (B-lactamase) and have it inactivate the antibiotic while being able to synthesize all other cellular components necessary to its growth. this is usually translated by a very small lag phase (and this can be practically used in transformation protocols : you can often just drop the recovery growth if ampicillin is your only selection agent).
- For kanamycin: that antibiotic gets in the cell, and tries to block protein production. Therefore, before a bacterium can do anything, it musts get rid (in this case inactivate) of the kanamycin. the lag phase in planctonic cultures (~45 minutes) is basically the time it takes for the bacterium to have degraded the antibiotic below the M.I.C. The same effect could happen on your plates causing the smaller colony size. Try growing your plates one hour or two more if you really need same-size colonies as for ampicillin (but then, mind the additional lag phase if you use the colony to inoculate liquid medium).
cheers
Sandeep: I too face the same experience.The reason might be antibiotic concentration..But you can do again plate from those tiny colonies, hopefully you will get good colonies.In Parallel, try to initiate small culture purification..if that works you can confirm your protein..
Recently I meet with a similar situation, I don't think the reason is antibody concentration because there are two different size colonies, after plasmid prepared and sequencing I find the bigs (colonies 1, 2, 3) are original vector, but the smalls (5, 6) are my expected recombination vectors.
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