Extraction of DNA from E coli
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- Опубликовано: 27 июл 2024
- Demonstration of the extraction of DNA from E.coli. Cells were harvested, pelleted and diluted in TE Buffer pH 8.0 [0.15 M NaCl; 1 mM EDTA; 10 mM Tris]. 1 mL of 10 mg/mL Lysozyme was added to break down the cell wall. 2 mL of 20 % (w/v) SDS was added to dissolve the exposed plasma membrane and lyse the cell. NaCl was added to a final concentration of 1 M, to dissociated any proteins bound to the bacterial DNA. An equal volume of Chloroform: Isoamyl Alcohol (24:1) was added to precipitate the proteins. Centrifuge separated the aqueous from the organic layer, with proteins either dissolved in the organic layer, or stuck between the two layer at the interphase. The upper aqueous layer, containing the DNA, was removed, leaving behind the protein. 100% Ethanol was added to a final concentration of 67% (v/v), to precipitate the DNA out of solution. A glass Pasteur pipette was used to bind the DNA and transfer it to a new test tube. Finally the DNA was dissolved in TE Buffer.
UV Spectrophotometric analysis resulted in an absorbance of 12.81 at 260 nm wavelength & 6.63 at 280 nm wavelength. The ratio of 260:280 of 1.93 indicates the DNA sample to no be contaminated with proteins. The 12.81 at 260 indicates the DNA concentration to be 640.5 ug/mL in 5 mL TE Buffer. 
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1000% best youtube video for instruction I have ever seen.
Thanks for your feedback. I hope you get a chance to isolate DNA in the future.
Very articulate and well demonstrated. Keep doing it Prof
Thank you, I will.
thank you very much mr drew...from algeria
Thank you sir, from India
this video really helped me understand the process thanks .
Happy to hear it increased your knowledge.
Jazak Allah sir. Its the best 👍🏻✨
Great work sir
Thank you
THANK YOU SO MUCH SIR
thank you sir
thanks for the information professor
You are welcome
THANK U SIR
Really Helpful Prof♥️
Glad to hear that.
Good
Thanks for your great work! In the lab session at my university, we often use sodium acetate to neutralize the charge of DNA, making it less polar and thus could be easily pulled out of the solution. I assume NaCl used in this video is also for the same purpose (along with dissociating DNA binding proteins). But in my case the salt is added later with alcohol in precipitation step and we also have to deal with salt contamination. I'm kinda confused with this. Is it because of the different protocols?
Sodium acetate (NaOAc) is a fine salt to use in DNA isolation. It will also change the polarity of your DNA to allow for ethanol precipitation. When are you adding in the NaOAc? I add in the NaCl before the chloroform step to dissociate the DNA binding proteins from the DNA.
Are you using SDS as your detergent? If you are working with samples that contain SDS, you will want to use NaCl. The NaCl keeps SDS soluble in ethanol. Without it, your SDS might precipitate out of solution along with your DNA after adding in your ethanol.
In the end, you will need to optimize your protocol for your starting sample and desired end product. Optimization is half the fun. Good luck.
Thanks for your detailed answer! We add NaOAc after the chloroform step, right before DNA is precipitated with isopropyl alcohol. I think the protocol we use was optimized before as it was put into the lab manual. But in my case, SDS is used along with Proteinase K, not NaCl.
Thanks for the video professor, in my lab protocol after adding the sodium acetate we add isopropanol, and after passing the dna to another tube we add ethanol to make it 'compact', I think. I was wondering what is the name of the method used? Because I thought it would be ethanol precipitation but actually I think isopropanol is being used for that purpose, would the technique be considered isopropanol precipitation?
Hi Leila. In order to precipitate the DNA out of solution, you need to change the polarity of the solvent. That is the purpose of adding in the alcohol, to change the polarity. In all my years of performing this protocol, I have always used Ethanol. As Isopropanol is also an alcohol, it should also change the polarity of the solvent, leading to the precipitation of DNA out of solution.
I investigated this a bit further after your question and this is what I found. It appears you can add less Isopropanol to obtain the same result, as DNA is less soluble in isopropanol, so this would be an advantage of doing an Isopropanol precipitation. If you have very little volume remaining in your test tube, you cannot add Ethanol, as you require much more. I just read that salts are less soluble in Isopropanol; therefore, salts might also be precipitated out of solution with your DNA. If so, the recommendation is to wash several times with Ethanol after your Isopropanol precipitation. With Ethanol precipitation the Ethanol should be keep cold. Ethanol is more volatile than Isopropanol; therefore, it will be faster to dry your DNA with an Ethanol precipitation. I have also read that Ethanol is best for small volumes of DNA, while Isopropanol is better for large ones. My work with DNA has usually been in small volume plasmid preps, so Ethanol precipitation appears to be best for that.
In short, both methods are used to obtain the same result. In the end, I advice you to run an experiment comparing both methods on your sample. Determine which one gives you the best outcome and use that method moving forward. Thanks for your comment and I hope this helps you move forward with your work.
Hi Sir! I would like to ask if we decided to experiment something related to E.coli, where can we get a sample of it?
I obtain all my cultures from www.atcc.org
Hi Sir, I would like to ask this will contaminate with RNA?
If you are concerned about RNA contamination, you can treat the sample with RNase.
Prof u didn't use isopropyl alcohol? Is it not necessary to use for precipitation of dna.?
Alcohol is used to change the polarity of the solution so that DNA will precipitate. We always use Ethanol for our precipitation.
@@ProfessorDrewCollop thanks for the info, love from India
Hi, how do I know the dna sample I have collected is chromosomal dna but not contaminated by plasmid dna ?
This method will isolate all the DNA from the cells. There is a different protocol to isolate only plasmid DNA, which usually involves a commercial kit and spin columns. I do not know a technique to only isolate the genomic DNA from the cells without the plasmid DNA. There is probably is a protocol, but I have never needed to do this personally. This is one thing that usually surprises people. When you work in science, you get really good at very specific techniques, but other techniques are a complete unknown. The good thing is, that learning new techniques is usually pretty easy, if you have a strong set of foundational skills. The issue is time. There are only so many hours in the lab and most of them are accounted for in your regular daily activities. If you have time, network with others in the lab and get them to show you what they specialize in. Hope you can find what you are looking for.
how many sodium chloride did you add to the solution??
Thanks for the question. The final mass of NaCl depends on the volume. You want to raise the final NaCl concentration to 1 M (mol/L). You can use the molar mass of NaCl (58.44 g/mol) and 1 mol/L to convert your volume (L) into grams using stoichiometric calculations. ? L x (1 mol/L) x (58.44 g/mol) = ? g
Why do we use E.coli for DNA extraction
Thanks for the question Maissa. E.coli is the bacteria of choice in the lab. It grows quickly & easily in the lab, we use it for cloning DNA and we fully understand it's genome. Hope this helps.
1:54 Why should we keep lysozyme cold?
Lysozyme is an protein enzyme. If it gets too warm the protein will denature (lose it's 3D shape). If this occurs, the function of the enzyme will be greatly reduced. We keep it cold, so we can continue to reuse it over many days or months. At 4'C (fridge) the enzyme activity is minimized and the protein is stabilized.
@@ProfessorDrewCollop thanks for answering
👋🏽 hey what’s the name of this method ?
Some call it a chloroform extraction.
Sir thank u, is it applies to every other bacteria
This is a standard protocol for DNA extraction. As a result, it will probably work on other bacterial species. That being stated, there are so many different bacterial species, you might need to optimize based on what the strain is your are working with. If you do a PubMed search, I am sure you can find the best protocol you require.
@@ProfessorDrewCollop thank you very much. I have extracted DNA using your protocol. I got pellets also. I am so happy. Once again thank you sir.
How culture you used for this pellet to start with?
Hi Garima. We have stock solutions of E.coli for our labs. To obtain enough cells to create a large pellet, I created 5 agar plates and streaked them using the hockey stick method. You place a drop of E.coli on your solidified agar plate. You then make a glass hockey stick by heating and bending a long tipped glass pipette. Dip the pipette in alcohol and flame it to sterilized. Use this sterilized hockey stick to gently spread the drop of E.coli over the entire surface of the plate. Incubate at 37'C for a few days. Don't think I have made a video of the different methods of plate streaking. The other method of plate streaking is for isolation of a single colony. Perhaps during the Fall semester I can make record this, if you would like.
Please what is the significance of the incubation process sir?
Thanks for the question Rukkie. Which incubation are your referring to? Can you specify which step you are asking about?
Please the incubation step that was done after the addition of the lysozyme. You used a water bath for that.
Before the addition of the SDS
And you incubated again after that what was the purpose, Sir?
Lysozyme is an enzyme and has evolved to work best at 37'C, your body temperature. This is why I incubated it at 37'C, to activate the enzyme. When activated, Lysozyme will breakdown the cell wall surrounding the bacteria.
It hurts to see that you lost 1 drop of precious DNA at 10:06.
Astute observation.
Excellent sir can send me protocol to my mail
I have updated the description to included the volumes and concentrations. You should be able to write your own protocol from this.