6 replies to this topic

[icecubes83]

Hi ,

I need help in confirming the results from transformation. I did a miniprep for E.coli with pBR322, pBR329 and pML21.  After isolating the purified plasmid, I did a Restriction digestion with EcoRI. I also perform an experiment for retransformation of plasmid into competent E.coli ED8654 and the culture was plated on agar plates containing: ampicillin, chloroamphenicol, kanamycin or tetracycline

The question is :
How do I prove the points that the plasmids carry genes coding for the various drug resistant phenotypes without doing DNA sequencing?

Thanks for your help in advance

[little mouse]

I'm not sure I understood well your question.
If the transformed bacteria grow on agar plates containing an antibiotic (and non transforming bacteria do not), then the plasmid must carry a gene coding for the antibiotic resistance.

[icecubes83]

Hi Little Mouse,

Yes . I understand your point . However the question is : " I would need to prove that the plasmid do contain the genes for this drug resistance phenotype by another experiment other than verifying the results using DNA sequencing ?


icecubes

little mouse, on Feb 2 2009, 09:44 AM, said:

I'm not sure I understood well your question.
If the transformed bacteria grow on agar plates containing an antibiotic (and non transforming bacteria do not), then the plasmid must carry a gene coding for the antibiotic resistance.

[little mouse]

Hmm,
make a western-blot to detect the expression of beta-lactamase?

[icecubes83]

beta - lactamase is for coding ampicillin right? then how do you do western blot to confirm the kanamycin, chloroamphenicol and tetracyclin encoding gene??

[little mouse]

icecubes83, on Feb 3 2009, 07:11 PM, said:

beta - lactamase is for coding ampicillin right? then how do you do western blot to confirm the kanamycin, chloroamphenicol and tetracyclin encoding gene??

beta-lactamase is for ampicillin.
for the others I don't remember the mechanism. For kanamycin I think it's something about nt5... try to google it.

[psygnosis]

You are asking others to give you a direct answer to questions from your practical homework. If I'm not wrong, you are an UWA student that currently taking the Molecular Biology module SCIE 3326. Please spend some effort to do your own research.