I work on antibiotic resistance genes in bacteria. Resistance genes present on plasmid. I extract DNA using boiling method. Does this method suitable to obtain plasmid DNA to study resistance gene. If it is not suitable, what is the best method to extract plasmid and bacterial DNA for my work?
If you are just amplifying the genes (and your bacteria is gram negative) then this should be fine. If you are using the plasmid itself then no it will not work and you need a specific plasmid extraction protocol. For multi copy plasmids you can use minipreps. if you plasmids are low copy number and large (more than 70kb-90kb) I suggest you use the protocol in the link or a modified BAC extraction protocol.
Article A Simple, Rapid Method for Extracting Large Plasmid DNA from Bacteria
Hanna Alalam
Many thanks for your answer.
I only detect the presence of resistance genes in gram negative bacteria
Do you mean boiling method is suitable to study resistance genes on plasmid in gram negative?
Will plasmid DNA be extracted by boiling method?
Yes boiling should be sufficient to release the DNA. However, the mixture might contain inhibitors therefore it will be difficult to distinguish between a true negative and a false negative therefore I suggest you run an additional reaction for each extraction you run using ribosomal primers just to make your preparation is clean enough for PCR.
Hanna Alalam
can you give me link for for additional step to make my preparation clean?
You can either go with a method that is a modified phenol chloroform extraction:
Article Extremely Rapid Extraction of DNA from Bacteria and Yeasts
or you can use a genomic DNA kit (spin columns):
example kit:
https://www.sigmaaldrich.com/catalog/product/SIGMA/NA2110?lang=en®ion=SE
it is up to you to decide what is optimal based on the species you work with. However, as I said before boiling should be sufficient if you run a positive control for each extraction.
Samah Mohammed Refay thank you for your question
Thank you for your answer Hanna Alalam , Best regards
Hanna Alalam is correct, the boiling method works fine for small plasmids, but I'm not sure it is the best approach for isolating large plasmids. If these are natural drug resistance R-plasmids, they are often quite large (30-150KB in size) and you may not get good results using the boiling method.
Michael J. Benedik
Drug resistance present on plasmid but i dont work on specified plasmid. I only detect the presence of gene in bacteria, does the boilig method is suitable?
If it is not suitable, what is the best method?
I would recommend you the Alkaline Lysis method for plasmid extraction. i never used boiling method for plasmid extraction. it can give u a sufficient amount of plasmid Dna. if you want an optimized protocol of alkaline lysis methods, let me know
Boiling method works quiet well as Hanna Alalam mentioned. Only one thing extra: boiling in 1x TE gives more DNA stability over time.
Sayed Abdullah
I need a protocol of alkaline lysis methods. Thanks alot for your respond and help.
Samah Mohammed Refay there seems to be a confusion in the answers provided above. You only need to do alkaline lysis if you want to extract intact plasmids without genomic DNA and what you want is just to do PCR, making methods that particularly extract plasmid sequences just an extra hassle. However, if you insist on doing alkaline lysis here is a link (however considering that most of the plasmids that carry the kind of resistance you are looking for are quite big, simple alkaline lysis will not be suitable to extract them).
https://bio-protocol.org/bio101/e30
Snap chill (Bioiling method ) is very good method for extraction of bacterial DNA. It gives gives good amount of qaulity DNA
Thanks
-Dhananjay
To the some limit it is ok.but due to presence of impurities it is not suitable. For that you can go phenol chloroform extraction method.
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