Unfortunately, in the complete absence information, the only way you will identify your plasmid is to sequence the whole thing working outwards from your gene  and venus fusion. There are just too many possibilities with REs and too many plasmids that use similar 'building blocks' (promoter, polyA, ori, etc.) to identify the plasmid you have.

The other option which may be simpler in the long term is to re-clone into a known vector, again you may have to do a little outward sequencing to identify RE sites or confirm the sequence at both ends of your fusion protein sequence to amplify out the whole thing by PCR.

Also get your whole lab to make a complete detailed inventory of every plasmid that arrives, source, sequence, anitbiotic resistance etc.. it sounds like a pain but in the long-term it will save the whole lab hours of detective work in the future. Your PI has no idea where it came from, no e-mail records or MTAs?

Unfortunately, in the complete absence information, the only way you will identify your plasmid is to sequence the whole thing working outwards from your gene  and venus fusion. There are just too many possibilities with REs and too many plasmids that use similar 'building blocks' (promoter, polyA, ori, etc.) to identify the plasmid you have.

The other option which may be simpler in the long term is to re-clone into a known vector, again you may have to do a little outward sequencing to identify RE sites or confirm the sequence at both ends of your fusion protein sequence to amplify out the whole thing by PCR.

Also get your whole lab to make a complete detailed inventory of every plasmid that arrives, source, sequence, anitbiotic resistance etc.. it sounds like a pain but in the long-term it will save the whole lab hours of detective work in the future. Your PI has no idea where it came from, no e-mail records or MTAs?

I agree with the above.  You might also contact former members of your lab or neighboring labs.  Someone will probably remember obtaining the plasmid.

Thanks for the responses. The former lab member who received the plasmid did not keep any records of source. I'm waiting to hear back from my PI on email records.

And unfortunately, this is only one of many plasmids I have had to do detective work on. I have been working on documenting all the plasmids because the record keeping has been completely atrocious. 

The real problem with this situation, apart from wasting valuable research time doing detective work, is that if you manage to generate data with these 'anonymous' plasmids I don't understand how you can cite them in your publications? This is something that your whole lab, including your PI, should really take more seriously.

Good luck with the mapping, if you have raw sequences (fasta or seq formats) then Snapgene is very good at automatically recognizing features (promoters etc.) to make presentable maps for your lab quite easily.

All good responses here. Just know that even IF a former labmate recalled the name and source of the plasmid, I would be wary anyway. I have received plasmids over the years from other labs that were not what was intended. I've had inserts in backwards, unwanted stop codons upstream of the ORF, and cross-contaminated plasmids. Coupled with Nick's good comment above regarding "anonymous" plasmids, I would follow the advice above of sequencing ends, subcloning into a known vector, and then sequencing the whole ORF. Or primer walk around the whole plasmid if there could be other features in it that you would want to maintain.

Hi Catherine,

if you are going to be looking at many different plasmids, you might want to design sequencing primers for the most common elements in plasmids. So that you can probe and sequence with them. Could provide a shortcut in the sequencing of the whole plasmid.

-Richard