E. coli transformation issues - old plasmids (Jun/18/2007 )
I've just started in a lab where I am told we have 3+ year old plasmids. Is it safe to use these, or should I run them on an agarose gel first? I haven't been able to find lab notes from the guy who made the plasmids, so I don't know anything about the inserts, other than that the various plasmids are mutants of my gene of interest. Do I need this information now? Can I get started with my transformation experiments, and if so, do I need to use a DH5alpha strain of E. coli to purify plasmids (since I don't know their condition), or should I jump straight to a BL21 strain for protein expression? Lots of questions...I'll take answers to any of them!
I think it depends on alot of things...
1) what are the storage conditions for the plasmids, I assume these were maxiprepped or something?
2) how much do you have left?
3) what experiment are you doing first?
If purified plasmids were stored at say -20 or -80 and you have plenty of the purified plasmid then first run gel to ensure are intact then send some small volume for sequencing and go ahead with protein expression you can get your expression data and match it up with the sequence analysis later and know what you have... (assuming the experiment is such that you can get the data in a "blinded" run...)
If plasmids are degraded on gel, you don't have enough, or they were stored improperly then do the DH5-alpha transformation first then purify (maxiprep etc) then continue...
Even if you can do your first experiment without knowing what each plasmid is exactly, you will eventually need to know what the inserts are so you will have to sequence or do digest or something to determine what each clone is no matter which approach you take...
HTH and good luck!
"Trust no one" if one may quote X-Files.
So true to these words, don't assume that the plasmid is correct. Currently the production notes concerning this plasmid are not avaliable (Can you find it?). No way to tell if the construction work was done correctly. Thus I would say you must sequence the gene you are working on. First find out how much DNA you have. If insufficient, make more. Then sequence said DNA. Check the sequence to what it should be. If correct go ahead and do the expression work.
1) what are the storage conditions for the plasmids, I assume these were maxiprepped or something?
2) how much do you have left?
3) what experiment are you doing first?
If purified plasmids were stored at say -20 or -80 and you have plenty of the purified plasmid then first run gel to ensure are intact then send some small volume for sequencing and go ahead with protein expression you can get your expression data and match it up with the sequence analysis later and know what you have... (assuming the experiment is such that you can get the data in a "blinded" run...)
If plasmids are degraded on gel, you don't have enough, or they were stored improperly then do the DH5-alpha transformation first then purify (maxiprep etc) then continue...
Even if you can do your first experiment without knowing what each plasmid is exactly, you will eventually need to know what the inserts are so you will have to sequence or do digest or something to determine what each clone is no matter which approach you take...
HTH and good luck!
HTH? I was so excited to see two responses- thank you so much! I know absolutely nothing about the plasmids and how they were prepped, or even how much is left. I found out today that I can call the guy who made the plasmids, so your response gives me some good questions to ask. If I have to purify the plasmid, the maxiprep is the kit from Qiagen, right? (I've been trying to learn the steps) Also, if the guy who made the plasmids can't tell me info. on the inserts (he should, though), do I use restriction endonucleases to digest? If so, how do I know which enzymes to use to cut plasmid if I don't know anything about it? Thanks for the advice.
Clueless...
So true to these words, don't assume that the plasmid is correct. Currently the production notes concerning this plasmid are not avaliable (Can you find it?). No way to tell if the construction work was done correctly. Thus I would say you must sequence the gene you are working on. First find out how much DNA you have. If insufficient, make more. Then sequence said DNA. Check the sequence to what it should be. If correct go ahead and do the expression work.
I agree with you. I don't trust the plasmids! How do I determine how much DNA I have? I know that we can cheaply send off plasmid to be sequenced in our own building, so that's pretty straightforward. Do I make more via DH5alpha transformations? Then purify via Qiagen maxiprep kit? (how do I know to use miniprep or maxiprep?) If sequence of DNA is correct, continue with BL21 transformation and purification? Thank you so much for the advice!
Clueless...
Yes, you do restriction endonuclease to conduct the restriction digest. Ah, but to use these enzymes you need to know the sequence of your plasmid. At least what the sequence of the plasmid should be in theory. If the pattern of DNA fragments from the cut plasmid matches to what you expect to see, then the plasmid may be correct. A restriction digest, only shows the general structure of the plasmid, not the fine scale detail.
If you can contact the guy who built them,
Try getting the below information
1.The nucleotide sequence of each plasmid. Annotated with all the features. If you can get the digital map, put said info in to a program like Vector NTI, useful program that helps display your plasmid. THis information is vital for later cloning work and restriction digest analysis.
2. How he actually built said plasmid. All the steps, he did in the cutting and pasting of DNA. Where he got the vector from, how he obtained the mutant genes. His note books, essentially. (in an ideal situation). Since these are mutants genes in a plasmid. Where are these mutations, the nature of the mutations. Which base pairs where changed. What is the name of the plasmid that he used to carry the mutant genes.
3. How did he confirm that his plasmid are correct? Did he do only restriction digest. (This only confirms that the plamid looks about right. But an error could still be in hiding) Did he do a sequencing, to confirm that every last basepair is correct. A single base pair change can prevent protein expression or give you errornous results. Can you have the sequence traces (ie the data from the sequencing reaction) for you to look at and keep.
In my book, a plasmid without all the above is not suited for use.
If you can not get (1), you can assemble the map using information from (2). If you have (1) you could live without (2). But you absolutely need (3). Mutations do sometime occur when making a plasmid. And human mistakes do on the rare occassion happen. You must be able to confirm that all is well, else you may be caught flat footed when your protein just doesn't work right.
Clueless...
Yes, you do restriction endonuclease to conduct the restriction digest. Ah, but to use these enzymes you need to know the sequence of your plasmid. At least what the sequence of the plasmid should be in theory. If the pattern of DNA fragments from the cut plasmid matches to what you expect to see, then the plasmid may be correct. A restriction digest, only shows the general structure of the plasmid, not the fine scale detail.
If you can contact the guy who built them,
Try getting the below information
1.The nucleotide sequence of each plasmid. Annotated with all the features. If you can get the digital map, put said info in to a program like Vector NTI, useful program that helps display your plasmid. THis information is vital for later cloning work and restriction digest analysis. Is NEBCutter a similar program?
2. How he actually built said plasmid. All the steps, he did in the cutting and pasting of DNA. Where he got the vector from, how he obtained the mutant genes. His note books, essentially. (in an ideal situation). Since these are mutants genes in a plasmid. Where are these mutations, the nature of the mutations. Which base pairs where changed. What is the name of the plasmid that he used to carry the mutant genes.
3. How did he confirm that his plasmid are correct? Did he do only restriction digest. (This only confirms that the plamid looks about right. But an error could still be in hiding) Did he do a sequencing, to confirm that every last basepair is correct. A single base pair change can prevent protein expression or give you errornous results. Can you have the sequence traces (ie the data from the sequencing reaction) for you to look at and keep.
In my book, a plasmid without all the above is not suited for use. I concur.
If you can not get (1), you can assemble the map using information from (2). If you have (1) you could live without (2). But you absolutely need (3). Mutations do sometime occur when making a plasmid. And human mistakes do on the rare occassion happen. You must be able to confirm that all is well, else you may be caught flat footed when your protein just doesn't work right.
This was so helpful! Thank you so much!!!
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This was so helpful! Thank you so much!!!
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you already got so much idea about it though i want to say it again Dont trust any plasmid