I have three Plasmids in glycerol stock. I labelled them and kept in -80 freezer for 6-7 months. Now when I wanted to take them out from the freezer I found the the labels disappeared. Now is there any way to identify them which plasmid is which?
First you can digest your plasmid samples with common restriction endonucleases ( you know which endonuclease is appropriate for your plasmids) and run on 1% agarose gel electrophoresis to check the size of your plasmid for correct identifiaction. Moreover, You can do PCR with plasmid sequencing primers and check the PCR product on 1% agarose gel to make sure your plasmid which one is which one. I think you can be able to identify you plasmids. TQ
First you can digest your plasmid samples with common restriction endonucleases ( you know which endonuclease is appropriate for your plasmids) and run on 1% agarose gel electrophoresis to check the size of your plasmid for correct identifiaction. Moreover, You can do PCR with plasmid sequencing primers and check the PCR product on 1% agarose gel to make sure your plasmid which one is which one. I think you can be able to identify you plasmids. TQ
best way to throw it. u can also grow the plasmid and check with the restriction digestion and sequencing to confirm it. This is a lesson for u. never forget to properly labelled on the tube
If you can grow your bacteria (I assume it is bacteria) then PCR reaction with specific primers (if you still have them) is a good start. Otherwise, sequencing should be the easy solution.
Better to through the culture and again transform it If you still want to check as your plasmids contains specific antibiotic selection markers grow the culture and plate in specific antibiotic medium for each selection marker that will also tells you if any of the plasmid has been lost.
I suggest doing mini-prep plasmid preparations from all 3. Digest each with restriction endonucleases used in their construction and check for the proper DNA fragments.
Hi,
Throwing them out is easier way but if you do not afford that option you can follow the following easy steps..
1. if the plasmids have different antibiotics resistance genes then it will be the easiest way to identify... streak the each of the plasmids on LB agar plates containing all three different antibiotics... each of the plasmid will grow in their respective antibiotic containing plates....
2. if all the three plasmids are of same antibiotic resistance genes..(as often the case is).. then you streak and do mini prep from the culture and do PCR with specific primers for your specific plasmids.
3. If you do not have specific primers for each plasmid, then you can search for the presence of unique restriction enzyme in each plasmid and do the restriction digestion...
I hope this will help.
Good luck :)
The simplest way will be to miniprep and then sequence using primers targeting the MCS on the plasmids. Of course the assumption is that you have some idea of what were the plasmids carrying.
If size of each plasmid is different, then go for miniprep and run the plasmids on agarose gel. you will come to know about your plasmids.
But if size of all the three plasmids are same, then as suggested by the above scientists, go for PCR and restriction digestion
So many answers. What is the status of antibiotic gene (s) in your plasmid? is that different one? If different, just grown on LB brother and let you know about plasmid.
If it is same, then grown them and go for the colony PCR for the respective gene. Otherwise, go for the restriction digestion as fellow researchers suggested here.
Thank to everyone again for your suggestions. My plasmids have the same antibiotic resistant gene. Now i am growing then in Agar, and will do the Midi prep.
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