Miniprep problem - (Dec/24/2005 )
when does a miniprep not work?
Hi! could you please explain your technique more thank you.
i am using a miniprep kit for isolating plasmid DNA from DH5alpha..... inspite of following the protocol (standardized).... i did not find any DNA when i ran the gel....i had dissolved the DNA in 75 microlitre of the eluting solution.... any suggestions? thanks
Did you do washing for the pellet with 70% or 75% ethanol?
i had transfered the lysate to a binding column provided by SIGMA and added the wash solution containing ethanol and then eluted the DNA with the elution solution.... still i dont understand how i lost the DNA...
Hi there, some questions that may answer yours
How much elute did you check on the gel? It may need more.
Was it a new transformation or an old stock? It may be that you got a contaminant that carries no plasmid.
Did you use the correct selection media? Some plasmids carry different selection markers.
Check if u have to precipitate the elute! (isopropanol and 70% etoh wash)
did you do the extra steps to check the wash fractions? some protocols (I am thinking of Qiagen because that is what I use) recommend pulling samples here and there and running them on a gel, just like with a protein purification, to see where you lost the sample
this could point to problems with the kit buffers, etc
perhaps your eluant has been contaminated with nucleases?
if your plasmid is low-copy, you may need to start with a higher volume culture or something...again, your kit instructions may have a protocol suited to this situation
we use the sigma miniprep kit in our lab routinely,
DH5alpha is a high copy number strain, so your plasmid should have no problem there.
There are troubleshooting steps included in the kit. One question, did you see any DNA in the wells of your gel? I am not too sure if you are using a high percentage gel and you didn't say if you digested it with a restriction enzyme, so it could be still in the gel as it is high molecular weight, however if you did digest it, you may have nuclease contamination in your restriction enzyme.
have you measured the DNA concentration on a spec? this will tell you if your eluate actully contains DNA
A standard problem is forgetting to add ethanol to the wash buffer, so that your DNA washes out of the column during the wash step. Smell it to check.
Another is to grow the overnight without antibiotic, losing the plasmid.
There is no such thing as a "high copy number strain." The copy number of the plasmid is determined largely by the plasmid sequence, not the E. coli strain. Your plasmid could be a low copy number plasmid, which would reduce dramatically the amount of DNA recovered. What is the plasmid backbone?