How to check for the presence of plasmid?
Posted 05 April 2013 - 10:10 AM
I am new to mol gen and I have been encourtering trobles with chemical transformation lately. However, after a couples trys, i finally got it to work, but I am not sure the colonies are what i think they are. So i am thinking that i should check the for the presence of the plasmid, to see if the colonies are what i think they are.
I am a bit stuck on how i should check for the plasmid, my understanding is that I inoculate the colonies in LB with appropiate antibiotics, then preform a mini prep to isolat the DNA, then run a gel to check for the presence.
I am not sure if that is the way.
Posted 05 April 2013 - 10:47 AM
But make sure the bacterium you use does not have a plasmid by itself allready.
When you put it on gel, you can see how big the band is on your gel, so you know whether it can be your plasmid or not.
You can also check it with RE (cutting the plasmid) if you know the sequence to check it again (and put the cutted plasmid on gel too)
If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.
Posted 10 April 2013 - 04:33 AM
PCR reaction follows the standard protocol:
5min heating to 95°C (where your bacteria gets lysed and DNA/plasmid is set free)
cycle 25-35 times with
30sec 56°C (dependent on primers)
30sec-2min 72°C (depends on amplicon size)
10minutes 72°C for final elongation, then do gel electrophoresis to check for correct amplicons. Colonys that show the right size can then be amplified and mini/maxi prepped.
Edited by 2xzwei, 10 April 2013 - 06:00 AM.