Dear all
I am facing a problem with plasmid recombination, even in Stbl3. The construct that I am trying to create is basically NFAT3x RE upstream to a GFP so as to make a NFAT-reporter cell line. Somehow, I have observed in as many as 16 colonies that I get after transformation of the ligation product, that 15 of them have got recombined. I have tried patching and growing the colonies @ 25degC for plasmid isolation, but still I am observing recombination.
I analyzed 3-4 colonies with digestion that suggested that the recombination is occuring in the region where I am trying to insert my clone (Basically the RE site flanks that region and the size of the region that I am getting is 700-800bp less than what one would expect in the parent vector, let alone the correctly ligated vector which only adds up to it).
Could it be that I even need to grow colonies @ 25degC post transformation of the ligation product ? What else can be done?
Some plasmids are difficult to clone because of the recombination you're experiencing. Growing bacteria at 25 deg C and choosing a mutant cell line like Stabl3 s a good strategy to minimize recombination.
I would address the problem by doing PCR screening of 40 or so colonies. The idea is to prepare a master mix (MM) and pipet to all tubes. Pick a colony with a pipet tip and scratch it against the tube containing MM. - In a separate LB plate with antibiotic streak the bacteria from the tip. Make sure the numbers match. Colonies can grow at room temperature.
Of the clones that were positive by PCR screening (one primer on the vector, another on the insert), grow minipreps or go directly to midiprep. Confirm which clones are correct by PCR, digestion or sequencing. Many of the initially positive clones will turn out to be negative or defective, but a few might be right.
Hope all of you are good.
First of all, Thanks @oscar for your comments.
I would share a good news with you. One of the colonies out of 16 that I had suspected to be positive indeed turned out to be positive.
Following seems to have worked for me in case of this unstable clone :
1. (Even though I did not try this) Ligation mix transformed bacteria should be plated and grown at 25degC.
2. Further patching of bacteria on the plates for colony PCR or something should be again plated and grown at 25degC.
3. For miniprep/midiprep, grow cultures at 25degC and slow RPM. For instance I grew 1-1mL cultures in 2mL MCTs @25deg and 100rpm. ( Horizontally placing the MCTs ). Similarly, I grew bacteria in 20mL media (in 50mL falcon) @25deg and 80rpm.
I hope this would also help other researchers facing similar problem in the future.
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