check plamid identity after miniprep - (Apr/08/2006 )
I transformed and purified 2 plasmids. The 2 plasmids only have 1bp difference, and the point mutation site is not a RE site. I did a restriction digest but all I can get from it is that the sizes of both plasmids are correct. So I don't know how can I check whether the 2 plasmids are not mixed up or not? One thing I can think of is PCR a 100-200bp fragment that include the point mutation and sequence the PCR product. But I know nothing about sequncing and wondering where I can get a protocol? Or is there other ways to check the plasmid identity out?
Sequencing would be my choice. Do you have a sequencing facility available to you? If not, manual sequencing would be (IMHO) too much work...
If you know the sequence of your insert and the basepair that has changed, you might be able to detect the difference with PCR by designing a set of two primers in which the variable base is the 3' base of the primers. Couple each of these with another oppositely oriented primer in a PCR reaction.
The 3' end of the primer is critical, thus (perhaps) the variable primer will only produce a product with the correct template, and fail when used with the other?
Thanks for your immediate reply, Homebrew.
I know every single base pair about my plasmids. The 3’ PCR primer is a good idea. But if you recommend sequencing I might try to give it a go.
Sequencing facility is not a problem. My problem is I don’t know how to use the machine. All I know is a bit Sanger sequencing theory I learned from undergraduate lectures, but that seems not gonna help in practice.
Can you explain a bit more about what product I need to prepare and what protocol I can follow for sequencing?
Iam not sure how good this technique is but i have come across people using plasmid DNA with single bp change in sscp gels which involves heat denaturation of your plasmid DNA snap cooling and electrophoresis in polyacrylamide gel. why dont you try it..
I have used PCR product to detect point mutations with single bp change in sscp gels which involves heat denaturation of your DNA and snap cooling and electrophoresis in polyacrylamide gel followed by silver staining of the gel. Run a control of your original sample also. If your test sample shows a shift compared to your control then you can be sure the the change in bp composition even a point mutation can be detected. you can try the same with your plasmid DNA too but you may have to linearize you DNA prior to it.
if you have a sequencing facility near you, ask them which protocols they'd prefer you to use......
generally, Applied Biosystems reagents and machines are used and the sequencing reaction is basically a pcr with labelled nucleotides (big dye terminator ready reaction mix). then you'll need to clean up your product; either with a standard sodium acetate/ethanol or isopropanol precipitation, or alternatively a kit eg. qiagen dyex kit.
it is pretty costly if this is just a one off
check out the website for more info