I am currently struggle with plasmid isolation from clinical drug-resistant bacteria. I am using simplified alkaline lysis:
(1.) Resuspension of the pellet in 50 mM glucose / 10 mM EDTA / 10 Tris-HCL
(2.) Lysis in 0.2M NaOH / 1% SDS
(3.) Neutralization with 7.5M ammonium acetate and chloroform
(4.) Precipitation in 30% polyethylene glycol / 1.5M NaCl
The main purpose is to (a.) determine plasmid size, (b.) prepare plasmid MLST, and (3.) confirm the drug-resistance gene presence on isolated plasmid.
I suppose that it would be reasonable to perform plasmid linearization prior to electrophoresis. Otherwise I should use a supercoiled molecular weight marker?
Summarizing, my main questions are:
1) Which restriction enzyme would be proper in this case?
2) I am not sure about possible gDNA / RNA contamination. Is this a contraindication for restrictive enzyme digestion?
Dear Piotr
- yes you need to linearise your plasmid for accurate size determination. Otherwise various conformers caused by supercausing tend to result in 2 different sized bands; neither of which are completely accurate in size
Laurence is correct that you need to linearize for accurate sizing. There is no way to predict which enzyme to use without knowing the sequence. What you might try is to digest your plasmid with a number of restriction enzymes and some subset of them should obviously linearize the plasmid. If you have a small amount of genomic DNA or RNA that should have no impact on the digestion or the gel.
An easier and accurate way is to perform S1-PFGE. S1 nuclease linearizes the plasmid and the run in a PFGE apparatus enables the estimation of both plasmid number and size.
thank you all for your advices. unfortunately I currently have no access to CHEF system, therefore I will try to apply 1D agarose electrophoresis.
I have attempted to linearize plasmids with EcoRI and XbaI. Below I upload a electrophoretic separation of 5 plasmid preps (uncut and RE digested).
Can it be assumed (for example) that XbaI-digested plasmid isolated from strain #3
a) the size of the plasmid is ~33kb?
b) and it consists of ~ 23kb and ~10kb fragments after digestion?
Does plasmid linearization is required for accurate plasmid size estimation?
Different forms of plasmid move with different speed on the gel (see attached figure). So, yes, you should use linearized plasmid to run the gel because the DNA size markers are linear form too.
Your another question: possible gDNA / RNA contamination.
If you have serious gDNA contamination, when digestion with a certain restriction enzyme, you might see some 'smear' on your lane. (similar to that from people is doing 'Southern' digestion).
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