I'm doing ligation (ligation high v.2) + transformation (ECOS DH5α) of quite a big plasmid (12.000bp, the fragment with the mutation is roughly 3000bp). I'm transforming 2 uL of ligation product in 25 uL of competent cells. I have colony growth but when I pick them up and resuspend them for mini prep I see no growth or the strange slime you can see in the attached picture. However, I have no problem when I transform the same WT plasmid from a older stock. Does anyone experienced this before and have any suggestion? Thanks.
Have you ever performed colony PCR screening before you pick colonies up?Because there are also too many vectors which are not cutted by enzyme.If you pick these colonies containing non-cutted vectors but recombinant plasmids,there will be no growth.
It is possible that the fragment you are cloning is toxic to the host cells. The slime looks like lysed bacteria. Does the fragment encode a protein? Is it being expressed constitutively? If so, you may need to use a tightly controlled promoter. I recommend the rhamnose or araBAD promoter, which can be tightly controlled. The lac or T7 promoter systems are too leaky for a toxic protein.
Are you using a 1:1000 dilution of AB in your liquid LB? Also, check the specifications of your vector. Some vectors can't handle inserts that big. It may become unstable and lose the insert. It is also possible that it is a problem with the DH5a stably maintaining the plasmid. When I did a larger vector or had large inserts I always used STBL4 instead of DH5a. STBL4s will take up and maintain large plasmids. Also, make sure that you transform and plate out cut and uncut destination vector without insert as well as the original vector that held insert, and cut and isolated insert; all as controls for your ligation. Just so you know exactly what is growing on your plates. Sometimes fragments will grow, or things will re-ligate and grow.
I agree with Cori. I had experienced similar problems with my plasmid which was about the same size (12.5kbp) with DH5a.
I found doing all my steps at 30 degrees rather than 37C solved the problem. This also dramatically reduced non-sense colonies as well.
Dear Melgari,
Your vector backbone has the antibiotic selection marker right? If you have added appropriate concentration of antibiotic to your plate, you should select the transformants. They might be negative clones i.e. carry only self ligated vector, but they should be resistant to the antibiotic. So, they should not have any problem in growing in presence of antibiotic in a liquid culture. If your protein is getting expressed in E. coli and it is toxic, colonies would not have appeared. So either there is a problem with your antibiotic or your vector (which carries the resistance gene).
1. Transform the comp cells with empty vector and see if they are having the same problem.
2. Check your antibiotic stock solution. If you are adding the antibiotic to heated agar, it might be losing its activity and hence whatever colonies you are getting are non transformants, later when you try to grow them in presence of antibiotic, since they have already grown without a selection pressure, they would have lost the plasmid by then and hence unable to grow properly.
3. Pick up the plasmid after 12 hours to avoid picking up spurious colonies.
4. When you inoculate in liquid media, take one with and one without antibiotic and see if there is any difference. Or else, take inoculum from the slimy growth and inoculate in media without antibiotic to check whether they are growing properly or not.
Hope it helps!
P.S. I wanted to ask Laumaea, whether it is specific for bigger plasmids only or growing at 30 rather than 37 is in general gives more positive colonies? It would depend on the antibiotic selection marker also I guess, so what is your selection?
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