in this video we'll talk about plasmid DNA isolation via the alkaline lysis method plasmid DNA is a really important tool in molecular biology lab people who are cloning the gene they need to propagate the gene in a bacteria by transformation reaction if you want to know more about transformation video is there in I button or description but anyway propagating the plasmid amplifying the plasmid or storing the plasmid is really important so overall in this video we'll try to understand how from bacterial cells one can chemically isolate the plasmid in a purified form so there are two methods by which one can do that there are column based purification which use the principle of affinity chromatography for purifying and there is also chemical purification which use fundamental uh chemical based approach to purify the plasmid obviously you can understand the first one is kid based and customized so this is more costly whereas the second chemical purification or alkaline lysis is an inexpensive method it's very cheap but very efficient so in this alkaline lysis method you start with solution number one in a moment I'll tell you the composition but you first resuspend the bacterial uh cells by resuspension buffer in the resuspension buffer you have Tris EDTA EDTA is very important because because it chillates divalent metal ions such as calcium and magnesium which are also important for dnases so by chelating these divalent ions you can also chillate DNA's activity also if you remove these calcium and magnesium it would destabilize the membranes as well glucose is also added in this solution to maintain the osmolarity and ensures the cells doesn't prematurely burst so overall the resuspension buffer has a composition like this so in order to prepare 100 ml of resuspension buffer or solution one you need to add all these components glucose Tricia EDTA water and have to adjust the volume till 100 ml then the important part is the alkaline lysis in order to take the plasmid out of the cell you have to realize the cell to take out the plasmid and this lysis step is performed by solution number two this solution like Number Two Can Be Imagined as a lysis buffer which contains sodium hydroxide and SDS so obviously by presence of sodium hydroxide you can understand this particular solution is highly alkaline and that gives us the name the alkaline lysis anyway the alkaline lysis step is performed by solution to whose composition is exactly mentioned here but I'll tell you what exactly happens after putting the alkaline lysis solution the solution to you mix it gently and wait a little bit of time such that the lysis can happen in this step first of all NaOH loosens up the cell wall SDS pops hole in the membrane and that releases the overall chromosome of bacteria and also the plasmid DNA in the solution now also the DNA gets denatured because all the interaction between the DNA is the base pairs the hydrogen bonds are now disrupted because there is an alteration in PH in the solution right and this is not favorable for hydrogen bond formation because it is highly alkaline so DNA gets denatured particular point of time if you take it um take the solution and open the tube you would see strand like appearances coming out of the tube now the next step is neutralization this extremely alkaline environment has to be neutralized and this neutralization is done by potassium acetate an acetic acid obviously by putting acid you can neutralize the base so it forms a salt known as potassium doticile sulfide sulfite with the SDS which is present in the alkaline lysis solution and this is an insoluble salt so after putting this solution three what happens is you see cardi precipitate like stuff appearing in the tube which are highly insoluble you can visually see them so then you wait sometime and then centrifuge them now once you add the neutralization buffer the waiting is important because you want to wait for precisely the time that would allow the circular DNA or the plasmid to denature but it doesn't give enough time for the denatured cellular DNA or the chromosomal DNA to denature so you have to ensure that okay renaturation happens only for the plasmic DNA so the timing is critical in this step after that one should one should centrifuge them and precipitate this insoluble substances the salt of doticile sulphate so after that what happens the aqueous portion is taken out and along with this aqueous portion 0.6 volume of isopropanol is added to precipitate the plasmic DNA which we which is supposed to be in this solution now isopropanol makes the DNA unhappy so isopropanol first uh performs bonds with the DNA DNA doesn't interact with the water so it's not any more solubilized it's now precipitated after ultimately it forms a pellet after isop isopropanol precipitation and this pellet can be washed again by uh ethanol washing so ethanol is added the precipitation is again done so ultimately after that the pellet is dried and resuspended in nucleus free water or let's say some Tris buffer this plasmid one once isolated can be stored in minus 80 or minus 20 degree Centigrade for years now how do you know the plasmid is good quality that you have isolated so you can run those plasmid in a gel and it should show you several bands I mean there are many outcomes possible like bands correspond to nicked plasmid linear plasmid super coil or circular closed circular plasmid the predominant form that you should be expecting is basically the super coiled one and that's why you should be getting the thickest band in the super coil region and other bands should be very faint anyway you can also check the quantity of plasmid that how much is the yield of this isolation process so one can put a one microliter plasmid there and ultimately one can check the absorbance at 260 by 218 nanometer it should come around like more than 500 nanograms per microliter for a good yield and a 260 280 ratio should be 1.8 for a pure DNA if this ratio is not proper then there could be a problem or contamination in your plasmic DNA by looking at these parameters you can understand how good the isolation is you can find more flashcards and notes in my Facebook Facebook page and you can find more notes in Instagram page anyway you can uh support the channel via super thanks you can use paytm PayPal or UPI for pain see you in next video