Does the bacterial DNA extraction by boiling method release plasmid DNA also?or is it just for chromosomal DNA?
Boiling bacterial cells lyses thel cell so it will release both plasmid and chromosomal DNA. If done properly with detergent and care then it can be used to selectively release plasmid DNA, it is one way of doing plasmid mini preps.
Thank you ....But i still have a doubt.....What if we resuspend the bacterial cell pellet in just autoclaved dh20 (without addditon of any other detergents) and place in boiling water bath for 10-15 min.....Will both plasmid and chromosomal DNA get released... And My organism is Vibrio....
Hi Reshma,
What I understand is that you are primarily going to do PCRs with the DNA isolated by boiling method. If this is true, boil samples in water for around 5 mins followed by high speed centrifugation and collect the supernatant, which is expected to contain both the plasmid and chromosomal DNA, suitable for amplification. However, if your target is to seperate the plasmid and chromosomal DNA fractions then boiling is not the proper method.
Just a practical advice: Try to use screw-cap tubes instead of eppendorfs for boiling.
SD
I agree with Sibnarayan's answer, if you just boil the cells then you will release both plasmid and chromosomal. I've never had a problem with eppendorf tubes though, you just need to put a weight on top or release the pressure after 30 seconds.
Thank you...My objective is to check for antibiotic resistant genes in Vibrio......I have isolated DNA by just boiling the pellet resuspended in water for ten minutes and used it as template....I got amplification in most of the isolates...My question is how to know whether this gene is plasmid coded or chromosomal borne..??
you will get both genomic and plasmid DNA but to identify gene you have to do PCR with specific primer and u can follow these ppl as they r working on cholera quite a long time. https://www.researchgate.net/profile/Bibhuti_Pal
https://www.researchgate.net/profile/Hemant_Khuntia/publications
I have performed PCR with antibiotic resistance gene primers and using DNA obtained from boiling method as template.... I detected presence of genes after PCR ....So now what I want to know is whether the gene is plasmid or chromosome coded....
Thanks a lot David sir for the detailed explanation and for the references provided....
Actually I have already performed plasmid profiling of the isolates and have detected the presence of higher mol weight plasmids i.e 33kb plasmids in my vibrio strains......But since the copy number of plasmids in vibrio is very less and bands were very faint I was not able to use it as template for PCR...
IS there any method to increase the plasmid copy number and band quality?
- Badges
- Science topic