Have you ever cloned a gram negative gene in a gram negative bacteria?I'm cloning two genes of a gram negative bacteria (thermophilus) in E.coli but after the cloning (positive pcr) and the growing up time,i do the miniprep and the plasmid never cut.I do the pcr again with the primers of the plasmid and the result is negative.Have you ever had the same problem?
Hi Carmela
I need more information about your experimental setup for be able to gave you a more preccise answer. Assuming that you are PCR amplifiing a Thermophilus gene and cloning it a in a suitable plasmid (Cloning method?), have you included the appropiate ligation and transformation controls?
Hi Carmeala,
These type of problems are often seen in cloning experiments. I'll suggest one thing
1) iif your first PCR check was from the very first plate...don't do this....instead pick the clones and patch them on another plate and then use this for the 'colony PCR'. The reason being the ligation mix used for transformation is also spread on the first plate and it may give you some false positive results...
2) some inserts are definitely unstable...but before concluding ...do your experiments with proper control...
bye
its not of incompatibility of gene and plasmid... rather spend more time in ligation during cloning and secondly plz select only transformed colonies..
I think you have problems with your vectors. u r using ta cloning vectors for ligation or normal re digestion vectors. improper digestion of of vector can sometime leads to like this. after u set up ur ligation, run 2-3 microlitre of mix in agarose gel to see shift in molecular weight. do fresh preps of vectors and re digestion. some how i am not too comfortable with colony pcr so prepare master plates n do minipreps.
Thank you all for answered!
In the meanwhile i've found (maybe) my positive colony before and after the miniprep and it'll go in sequence next week.Unfortunately,the other gene of the T.T. that i'm cloning too gave a lot number of colonies,but all the white colonies are negative(the screening is blue/white).I've modified the insert-vector ratio more and more times;however,I'm using a kit of the Stratagene,blunt cloning. Is a subcloning vector,not of expression. I'll try your advices,hoping for the best results :) Thank you all!
Carmen
i would suggest dephosphorylate ur vector, increase insert concentration and increase incubation time. what i do is in a small room i control the temperature of the room with ac to around 22-25C and there i do ligation for 2-3 hrs. n finally electroporation. sometimes all white colonies can also give u insert in wrong orientation. be patient. once i had screened 100 colonies to find none harbored gene in right orientation. good luck.
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