Welcome to today’s lecture, we are going to talk about DNA tools and gene cloning,so in the previous lecture, you have learnt about the theoretical concepts of DNA cloning,today, you will get familiar with the various steps, which are involved in performing DNAcloning in laboratory setup, you will also see that how a gene of interest is insertedin a vector then it is selected based on the presence of an antibiotic gene.So, let us start the lab demonstration session now, hello everybody today we are going todiscuss about the workflow of molecular cloning.Molecular cloning has some basic steps which we are going to go one by one, so firstlywhat we are trying to do is; introduce a foreign gene into an organism which we normally referredto as an insert, so this is my insert sample.So, what we do is; we take our insert DNA, we introduce into a carrier molecule normallyknown as vector, so most commonly use vectors includes plasmids, sot in this case we areusing a plasmid DNA in which we are going to introduce a foreign gene; our insert, sothere are as we may know plasmids, they occur naturally in bacteria’s and over the yearsstudies have been done, and you have seen that these plasmids also have some antibioticresistance gene.So, which confers a resistance against particular antibiotic to the bacteria, so if a bacteriais grown under that antibiotic condition, it will, it will; its growth will not be affectedand it will grow perfectly normal.So, here we have a vector DNA, we have our insert, so in order to introduce my insertinto the vector, so let us begin with the experiment, we have taken a vector DNA also,we have an insert DNA.What we are going to do is; we are going to treat them with restriction enzymes, restrictionenzymes are going to cut these DNA and they will create compatible ends, so that the insertand the vector they can join and form a perfectly circular structure.So, we are going to carry out the restriction digestion separately for insert a vector,so what we need to do is; first we take a vector, then we add the restriction enzymebuffer which is needed by the enzyme to carry out the digestion.We also add water to make up the volume and in the last step, we add the enzyme, so enzymeusually is added in a very small quantity, so I will use a different pipette for thatso, in this case we are using EcoR1 enzyme, so throughout the process we need to makesure that it is carried out using aseptic conditions, so the tip should we autoclaved,tubes also should be sterile, at the same time we should carry out the entire reactionon ice.So, right now I have made the cocktail; restriction digestion cocktail for the vector, I am goingto do the same thing for the insert and then we will keep the tubes at 37 degree whichis the optimum temperature for the enzymes to act.So, right now I am carrying a restriction digestion for the insert, so this is the insert,I am going to add the restriction buffer, so I am adding different volumes for eachof the components.And accordingly, I am adjusting the pipette as well, now I am adding water, so the lastthing is the enzyme, so I am going to vortex the tubes, so that the components are mixedproperly, then I am going to give them a short spin, so that whatever has stuck to the wallswill come down.Now, we will incubate the tubes at 37 degree for 1 hour.Now we will incubate the tubes at 37 degree.So, the incubation will be done for 1 hour, so after restriction, digestion has been performed.The next step is ligation of the 2 DNA’s, so we have our vector DNA as well as the insertDNA which has been cleaved by the restriction enzyme, so now we will join the 2 compatibleends using the ligase enzyme.So, here we have all the components of the reaction, first we will take rDNA, vectorDNA, then we will add the insert, so once we have added vector as well as insert, wewill add ligation buffer, so before adding anything, so we just need to pipette it alittle, you need to mix it a little bit.So, this is the ligation buffer, I will give it a small vortex, I will add this to theligation makes now, the last thing to be added is the ligase enzyme again, we are using asmall pipette because we require very little quantity of the enzyme, I will give it a shortvortex again to the enzyme, now our ligation mix is ready, we will vortex it a little,followed by a short spin.Now, we will incubate this at room temperature for 3 hours.After ligation is done, the next step is transformation, so after ligation the vector and the insertthey form a covalently closed structure and this DNA molecule, the combined DNA moleculewe introduce into a host cell, so in this case you have taken DH5 alpha host cell, whichdoes not have any restriction enzyme, which will chop of any foreign DNA, so we will introduceour DNA into this host cell, so that it can propagate further.So, this step transformation is carried out inside the laminar hood under a sterile conditions,so inside the hood, we have sterile filters, which may keep the inside air clean.So, before we start with the experiment, first we switch on the UV for 10 minutes, this stepensures that inside the conditions are extremely sterile, this step ensures that inside thelaminar hood conditions are extremely sterile under good for a work.So, after this we will switch off the UV and we switch on the fan and the light button.Now, we will carry out the transmission experiment, so we have our ligated sample, we will alsokeep a negative control water, so in all the experiments, we normally keep a negative control,so we have a competent cells which are the host cells in which we will introduce ourrecombinant molecule, in one of the tubes, we will add our recombinant molecule, in theother tube, we will add a negative control, water.This is our ligation mix which we will add in the competence cells, so now we will incubatethe tubes on ice for 30 minutes.Now, we will give the samples, heat shock treatment, so we will keep tubes at 42 degreeCelsius for 2 minutes.This heat shock treatment helps the host cell to get the foreign DNA; this heat shock treatmenthelps the foreign DNA to get introduced inside the host cell.So, now we will perform the plating, so as we can see, we already have plates; LB agarplates.These plates, they have a lot of components, which will help the bacteria to grow additionally,we have added the antibiotic ampicillin because a vector, the plasmid DNA has ampicillin resistantgene, so that when we will plate our ligated product onto this, only those colonies willshow up which have our vector.So, for 1 plate, I will be using the ligated sample which has been transformed into theDH5 alpha host cell.For the other plate, I will use the negative control which has been given the same treatmentas our ligated product, so I will take the sample, so I will add the sample in drops,so this is the spreader which I am going to use for plating, so this spreader is alreadydipped in ethanol, so that all the microorganisms if any will get killed, so I will put it toflame, so that the alcohol evaporates, I will wait for the spreader, spreader’s temperatureto get down.Because if it is too hot, it may kill my DNA sample as well, I can touch it here, so thatit becomes cool now, I will start plating, so I will be doing the plating in circularmotions, so that the sample is spreaded uniformly, I will do it till a point where I feel thatthe entire sample is spread uniformly and when the agar seems to look a little dry andthere is some friction at that point I will stop.One needs to ensure that we do not presser too hard else we may end up breaking the agarplate as well, I will show the lid some flame and I will keep it back, I will label my plate.Next, I will plate the negative control sample which is nothing but water, so normally wekeep the plates this way upside down otherwise, if we keep them like this, they sometimesbecause of the loose ends, we may end up having some sort of contamination, so usually wekeep upside down.Right now, I have done plating, so for some time I will keep it like this, straight way,later I will keep it upside down as well.Now, I will take my; now I will take the negative control sample again, I will add in the formof little drops, spread it uniformly, I will show my spreader flame, I will wait for fewseconds for the spreader, for the spreader’s temperature to come down, this is how my platelooks like.Now, I have started feeling some friction on the plate, so if I poke it further, itmay break, so at this point, I will stop, now I will take my plates and I will keepthem in the incubator.So, these are the plates which I will keep in the incubator for overnight incubation,so as you can see the temperature ranges from 37 to 38 which is ideal for the bacteria togrow.I can show you another plate, which we have already done transformation yesterday andhow a transform plate looks like.So, this is a transform plate which has a lot of colonies inside, so this in this caseof transformation efficiency has been very good.In some cases, we do not get as many colonies, so right now, we have kept 2 plates; in one,we hope that we have a good transformation efficiency and in the second plate, we shouldexpected to be blind because at it is just water to ensure that our experiments are absolutelyfine and there is no contamination.So, once you have obtained the colonies we need to be sure whether these colonies reflectedto recombinant molecule because as you know once we have done restriction digestion, wecan get different kinds of products.It may happen that during ligation, the vector itself ligates and have a self ligated moleculeor it may happen that there is only the insert that has transformed into the host cell, sowe need to be sure whether these colonies actually have a recombinant molecule, so onething we are sure that there is no insert here because the vector has antibiotic resistanceseen and here, in the media we have an antibiotic ampicillin.So, only those colonies which have the vector will grow on these, now those colonies canbe the self ligated vector or they can be the recombinant molecule having the insert,for that we will take these colonies, so for that we will take these colonies, we willgrow them, we will isolate the plasmid and we will check their size, so this is the LBmedia, this is the same media which has been, which is already you know in the solidifiedform in this agar plate.So, this LB media has all the components, which is required by the bacteria to grow,so we will pick up a colony, we will added in these LB media, we will also add our antibiotic,the antibiotic for which the resistant gene is already present on the vector, so thatonly our vector grows, no other contamination is seen.So, we will first add the antibiotic; this is our antibiotic ampicillin, everything weare doing near the flame, so that there is no contamination, there is no chance of anycontamination now, I will pick up a colony, this again we will incubate at 37 for overnightalmost 16 hours.So, this is the shaking incubator in which I will keep this tube for overnight incubation.So, you can see the temperature set is 37 which are ideal for the bacterial growth.So, this is how a bacteria culture looks like, you can see it is very turbid and after thisis a sign that the bacteria has grown properly overnight, so this is the tube which we havejust kept for the overnight incubation, you case see the colour of the tube, it is, itis not so turbid, it is transparent but after the grow this is how it looks like.So, in conclusion, I hope now your concepts for the gene cloning is much better and muchclearer now.As I have just seen the lab demonstration session with the advent of technological advancement,the genetic engineering can be performed in a laboratory set up with great ease now, whyit might you know look a straightforward but the cloning a gene often involves you knowlot of thought process and the whole parameter optimisation may take several months timeto really obtain the correct clone.And then you have to ensure that you know the sequence is correct and it is not self-ligatedvector, what you obtain is the right gene of interest which you started with, so ofcourse, the process is straightforward and much simpler but to actually obtain a rightclone in frame does need you know good listener operating protocols and sometime you knowgood experience of knowing the concepts involved in genetic engineering.So, I hope you enjoyed the lab session and we will continue our interactions and discussionabout gene cloning in the next lecture as well, thank you.