I want to extract plasmid dna from bacterial culture guide me what will be the most appropriate method.
Hi,
You may use either of two methods: commercially available kits or manual method, where you would need to prepare reagents yourself. You may search for alkaline lysis method for plasmid isolation, which is easy to perform and gives good results.
Good luck!
Hi Om Raj devi Sonia
One colony of recombinant E. coli was inoculated in 1.5 ml of liquid LB including Peptone 1% (w/v); yeast extract 0.5 % (w/v); 1 % NaCl (w/v); ampicilin 100 µgml-1 at 37°C overnight. After that, 1.5 ml of inocolumn media was centrifuged at 12000 rpm in 1 minute, harvest the sediment. 150 µl of solution I including of glucose 50 mM; 25 mM Tris-HCl; EDTA 10 mM; pH 8.0 was added and mixed by vortex. After that, 150 µl of solution II including of NaOH 0.2 M; sodium dodecyl sulfate-SDS 1% (w/v) was placed into that eppendorf and invert the tube gently. The sample was thawed in the ice for 30 minutes, then adding 150 µl solution III composed of CH3COOK 60% (v/v); CH3COOH 11.5% (v/v); H2O 28.5% (v/v), and mix well. Pipetting 450 µl chloroform : isoamyl alcohol (24:1) (v/v), mixed well and centrifuged at 12000 rpm in 10 minutes. 400 µl of upper phase supernatant was transferred to a new eppendorf, then supplemented by 320 µl isopropanol (10:8) (v/v). The sample was mixed well, and incubated at -20°C in 1 to 3 hours or at -84°C in 5 minutes. The samples were centrifuged for 12000 rpm in 15 minutes, discard the supernatant. After that, it was washed by 500 µl ethanol 70%. The tube was centrifuged for 12000 rpm in 1 minutes for harvesting the sediments and drying. At the last step, it was resuspended by 20 µl TE (Tris-HCl 10 mM; EDTA 1 mM; pH 8.0) plus RNAse (1%) (v/v) (Sambrook and Russel, 2001). 3-5 µl of product was tested on DNA electrophoresis.
if you want to use the plasmid for transfection, the purity and quality of the plasmid DNA are critical for successful transfection. Contaminants kill the cells and salts interfere with transfection. So, use commercially available plasmid isolation kits. If you are isolating plasmid to transform into other bacterial strains, follow Nguyen Tien Cuong protocol.
The plasmid preparation kits available from various manufacturer's can be used for best results else you can also go for manual methods for plasmid extraction.
Save money and time and go with the commercially avaliable kits. In addition , as Balamurali Krishna Vasamsetti said , the quality of isolated plasmid DNA is high and therfore it is suitable for various downstream applications such as sequecning, transfection and restriction digest.
Hi.
As Bhuwan Bhaskar said, the most common methods are two: manual purifications or commercial kits. I have been using both, and I strongly recommend you to use MIDIprep plasmid purification (in my case, I first use MINIprep purification for checking if I am working with the correct plasmid, and then I escalate it up to 200mL liquid LB medium, which I then use for MIDIprep purification). This method gives high purity and concentration, and it is relatively fast and clean. On the other hand, manual methods (alkaline lysis) has given me worsts results to date: if you load the resulting plasmid in an agarose electrophoresis gel, you will observe a smear following your circularized plasmid (with a low concentration), which is a clue of a low-quality purification.
Commercial kits for extraction are good, but where the resources are low u can use boiling lysis method which have been proven to give good quality DNA.
I have been using the boiling lysis method from bacterial culture and it has always been yielding good result, saving a lot of time and money.
You can use Qiagen plasmid DNA extraction kit, that gives the best result without any inhibitors
There is no single best method for plasmid DNA extraction. It depends on the size and copy number of the plasmid. If your plasmid is a small, high copy number plasmid such as a cloning vector, extraction is easy and almost any method will work. If you are working with a big, low copy plasmid, such as the F plasmid of E. coli, you will need special methods. In addition, the bacterial species plays a role. Some, such as E. coli are easy to lyse; others, like Staphylococcus epidermidis are very resistant to lysis.
So: What bacterial species; how big is the plasmid?
Andrew Jenkins Do you recommend which method for extracting large plasmid with few copies in E. coli? I have used alkaline lysis but sometimes the plasmids are not detectable. Also, I used commercial kits but I had poor results.
No, I don't think alkaline lysis will work well for large, low copy number plasmids. It's quite a harsh method and I suspect the cells burst quite violently, which would shear the plasmid DNA.
It's better to lyse the cells gently, using EDTA, lysozyme and Triton X-100, centrifuge out the cell debris and then concentrate the plasmid DNA, for instance by PEG precipitation. I remember getting nice results by binding the plasmid to a shallow bed of hydroxylapatite and then eluting it, but this method is said to be very dependent on the batch of hydroxylapatite and I don't recall the reference.
If you have access to an ultracentrifuge, the old Caesium chloride/ethidium bromide method is a good alternative.
Andrew
You can use a Qiagen plasmid DNA extraction kit or Monarch plasmid DNA extraction kit from New England Biolabs. I have used both and are very good
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts