Hello everyone,
In my case, i ligated my insert (804 bp) into pET30a expression vector (5422bp) 3:1 ratio containing 20 ul reaction by using T4 DNA ligase and incubated at 24 degree centigrade for 4 hour then transformed into E. coli DH5 alpha competent cell incubate 37 degree centigrade . Then i got colonies in transformed plate after that i steak those colonies in new fresh LB agar plate containing 50 ug/ml kanamycin after incubation at 37 degree centigrade then i did colony PCR of those steak colonies then i got positive clones in colony PCR after that i raised primary cell culture from the positive clone whatever i got positive in colony PCR and incubated at 37 degree centigrade for 16 h then i Shaw the culture it get turbid from this culture i am not able to isolate the plasmid I thought primary culture get contamination . what are the possible reason of contamination
can you me possible suggestion
what should i do for it
Hello,
To be sure of what is happening, do you have the controls:
1) make a culture of the bacteria not transformed by the plasmid, on the same medium containing the antibiotic and see if it grows, normally not.
2) Take the colonies of the non-transformed bacteria and do a PCR on the colonies, see if you have an amplification, normally not.
If it is 2 controls are negative, it means that the problem is elsewhere. Maybe the extraction of the plasmid was not good or the host bacterium rejected your plasmid
Did you include antibiotic in your liquid culture before plasmid extraction? Have you done a control PCR using the plasmid without an insert? What protocol are you using for plasmid extraction? What volume of liquid culture are you processing?
Dear Vishnu Dutt
did you perform all the steps of growth (in agar plate as well as in liquid in presence of kanamicin 50mg/l)?
Kanamicyn is a quite resistant antibody and it is strange that you carry up a contamination.
However i would like to suggest for next time to peak and perform PCR screening directly from the colonies obtained from the original plate. it is quite simple you can just touch the coloum with an inoculating nedle, strick in a twin plate to generate the and replicate and deep directy the same needle inside the PCR reaction.
In this way after the result of the PCR you can use the fresh duplicate to do an O/N inocula in LB+kanamicin and proceed to glycerol stock preparation and plasmid extraction.
In the following link you can find and outine of this process
https://www.blogger.com/video.g?token=AD6v5dzrZXbjCrsrnUvj5lmNOcOD_G10hIZc21CiOmmZoA6maPuNq-rPahUPP6mkAmCN3gEo8TW8PYjV4y-HkExg9cZ0gYqMyUGgoMr45rgDOWushBW8LocpxX5tGVjD633sQB3lRuVe
good luck
Manuele
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