I have problems with the recombination reaction of LR. I am starting building in pDONR221 (kanamycin resistance) and I want to recombine pYFC43 and pYFN43 vectors (kanamycin resistance). The solution that I have to this problem is given by strain selection (ie in agrobacterium) because pyfN43 and pYFC43 have pVS1 which can replicate in Agrobacterium. The problem is that when I do the reaction of recombination and positive colonies I am not getting transformation. Does anyone have suggestions to not get colonies?
hello, as far as i understand your problem is that you have the same antibiotic selection markers in both vectors, am i right? i had this problem as well in the past before i got pDONR207 in hand which has a gentamycine resistance. but before that i used to digest the entry clone with NruI e.g. that cuts in the kanamycine resistance gene in pDONR221. of course you have to check wether it cuts in your insert, if so you have to look for another restriction site. after digestion i used to separate linearised and non-cutted plasmid-dna by electrophoreses and performed than the lr reaction with the linearised entry clone. after transformation only successfull recombinated clones in your destination vector should be selected because the linearised entry clone is suitable for recombination but not for growth. i also read that the digest can be performed after lr-reaction if the enzyme does not cut your destination vector. however, with both variants you sometimes might still have to check some more selected but it worked for me. anyway the easiest way would be to work with pDONR207.
good luck!
Hi Maxi, thanks for the recommendation, I will linearize the vector as you say.
thanks
Hi Maxi, one more question, should I cut resistance or can be anywhere in the vector.
thanks
Dear Viera,
As per my suggestion, After LR reaction you should do transformation in E.coli selcted strain like TOP10. Varify by colony PCR using gene specific primers of your gene of insert. Then do plamid isolation of positive one. Check by PCR using plasmid miniprep as template. If it works well then you can transform in Agrobacterium. Some times the copy number is very less then colony PCR does not work as well and sometimes due to some unknown reasons.
Good luck
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