 |
what is the problem with inserts ? |
After cloning the insert of interest into LUXCDBAE plasmid, I got colonies in the antibiotic plates much more the control one , I isolate it in another plate and perform PCR using the same primers that I used to PCR the inserts for cloning , and I screened those new clones for Luminescence , the results of both PCR and luminescence was ok
But when I started the real experiment I had fluctuation in the results : the same colony which has been marked as good one , gave high luminance in the first trial but in the next day experiment under the same condition and using the same inducer it gave no tumescence at all , although the insert of interest has been confirmed by PCR using the same primers that I used for PCR of the inserts before cloning
take in consideration that I used new inducer and the concentration is ok
To enable tagging you will need to register on Bitesize Bio. We're sorry for the inconvenience, but it's free, only takes a few seconds, and it will enable you to view our seminars for free, ask questions from the professional community, and take part in the lively community of Bitesize Bio
| Strange cloning problem... |
|
To leave a reply you now have to register on Bitesize Bio. We're sorry for the inconvenience, but it's free, only takes a few seconds, and it will enable you to view our seminars for free, ask questions from the professional community, and take part in the lively community of Bitesize Bio
Nura Abboud
I am having argument with my advisor and I need your advise
Is it possible to use the phage contaminated strains for molecular bacteriolog y ? even after long time of the first contamination ? what is the best way to start experiments to avoid this phage contamination ?
I think that it is impossible to use those strains again as the phage may cause mutation and give false results while my advisor is insisting that I have to use the same strains !
SexComb
Is the antibiotic all right? Have yout tried to isolate the plasmid by alcalic lysis? Why do you use the cloning product directly?
It would be wise to isolate the plasmid DNA from a couple of colonies, sheck them by restriction endonuclease digestion, and sequence two of them. This way you would be absolutely sure, what you are working with, and it’s much more cost effective to transform bacterial cells with a palsmid than to clone an insert repeatedly. Not to mention, that this way you eleminate all errors, as you know for sure what plasmid you are working with, every colony would be the same.