I have done a lots of colony PCR recently but all of them do not show band after amplification, so I wonder is it because I didn't subculture them after transformation (keep for quite long). May I know whether this will cause plasmid loss and how can I confirm it?
Normally, the usual multicopy,colE1-derived plasmids that are used for cloning are not easily lost. You will see this if you try to cure such plasmids. This can be a nasty job sometimes. Will you perhaps give a few more details? What type of plasmid do you use? MiniF and pACYC derivatives are sometimes lost during storage, but rarely in all of your colonies.. Do you still see the phenotype due to the selective markers used for transformation? Are you sure, by control experiments, that your PCR conditions are alright?
Best wishes - Johannes.
Yup, my control in PCR is positive. The plasmid that I used is pJAZZ-OC Blunt Vector from Lucigen, it is used for metagenomic library construction.
Thank you. This is a linear type of plasmid. Unfortunately, I have no personal expertise with these molecules. I'm sorry. No helpful idea for the moment.
.... Maybe the distance between the primers is too long, because the binding sites are flanking the inserts?
If you are keeping the transformation plates too long, and directly work on them, you might be loosing the vector, because the ampiciline titer (or whatever selecting agent you are using) goes down, so there is no pressure for keeping the plasmid! You need to perform the colony PCR the day after transformation and as soon as you get your results, get your colonies out of the plate (subculturing or freezing them depending on what you usually do or need). I hope it helps you!
If your plates are more than 2 weeks old, you can throw them out, because your chance of finding viable colonies is quite low. For longer term storage, you should be either making stab cultures, or -80C glycerol stocks. Hopefully you still have some of your transformation/ligation mix that you can go back to, and pick up your cloning at the transformation step.
Oh my god...Its a bit impossible for me to finish colony PCR right after transformation because I need to screen for 2000 clones. Any idea what should I do?
Pick a single colony and dissolve it in 50ul of sterile water. From this you can do PCR using 5 ul and remaining amount can be used for inoculation. You can store remaining amount in 4C for a week ( I use it with in one week for inoculation). I hope it helps you..
I usually screen, using colony PCR, between 10 and 20 colonies, within the first two days after transformation
I would suggest you look for protocols developed for library generation that would facilitate handling and screening thousands of plasmids/colonies. One option would be to perform replica plating each week to maintain your bacterial colonies until you have finished your screening. Perhaps other simple approaches may help you out as well.
If I still able to grow my colony from the transformation plate, is this means that they are still okay to be used?
Yes, if your colonies still grow in liquid culture with the appropriate antibiotic, then your plates aren't too old for the PCR colony screening. Are any of your colony reactions positive? What is the size of the amplicon?
The size is about 500bp. I am doing screening for 2000+ clones and recently about 700+ not showing positive result.
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