How to check for the presence of plasmid? - (Apr/05/2013 )
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.
Yes, thats how you can do it.
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 want to screen a large amount of colonies so instead of miniprep you can also do a colony PCR on a single colony. Just pick it from plate and use an aliquot for colony PCR with primers that are able to identify your plasmid uniquely. (I always resuspend the colony in 5µL dH2O, use 2.5µL for PCR reaction and with the other 2.5µL you can go for miniprep if it turns out that your colony is the one you need, store it at 4°C for 1-2 days or freeze).
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.