[Music] hi everybody welcome back to synthetic biology one today I will be doing the ligation and transformation protocol so I will be like eating the RFP that we digested into the bio brick factor the total volume of the ligation reaction will be 10 microliters so first I have to decide how much volume of the vector and the insert I will put after calculating the concentrations and taking into account the length of the insert and the vector I will put 1 microliter of the vector and 3 microliters of the insert to keep the 1 2 3
ratio in terms of moles between the vector and the insert so here will be the tube for the legation and a good thing when doing a ligation is to also do a negative control where you only put the vector and no insert then after transformation you will be sure then for the positive sample the colonies you will see actually corresponds to your ligation and not to the relegated vector so first I will put 1 microliter of vector into both tubes so in the positive sample and also into the negative control and next I will put
three microliters off insert into the sample into the sample tube [Music] and for the negative control I will put three microliters of water okay also in the ligation mix there will be the ligase and the ligation buffer the ligation buffer has ATP and magnesium chloride which are both required for the functionality of the ligase the buffer it is 10 times concentrated so I'll put one microliter of buffer into each tube one and one the total volume of the erection will be 10 microliters as I said so we will have to add 4 microliters of water
into each tube [Music] and it's very important whenever you doing a reaction with an enzyme to put the enzyme in the very end once once there's already the buffer and other components in the mix so that the enzyme does not lose its efficiency so I will put one microliter of ligase into each of the tubes okay and now I will mix gently by pipetting up and down okay so now the mix is all ready and it's time to incubate the protocols for incubation can differ for most efficiency you can leave the tubes at 16 degrees
overnight but if you're in more of a hurry you can leave them for one or two hours at room temperature [Music] once the incubation is over I can now proceed to transformations so I will aliquot my competent cells into two tubes one for the negative control and one for our sample the cells are already thought and now I can add the ligation mix to each of the samples I will add one microliter but you can go up to 5 microliters total so 1 microliter of of the sample and 1 microliter of the negative control very
importantly you can flick the tubes to mix the samples but do not vortex them because otherwise it can damage the cells for maximum efficiency you can leave the cells on ice for up to 30 minutes but if you're in more in a hurry once again even 5 minutes will do alright now that the cells have incubated with the DNA I can now do the heat shock 42 degrees for 30 seconds okay the time is over and I can add medium to the cells to help them recover some people add 100 microliters but I prefer to
put one milliliter of medium to help them recover you can use lb or other regular media that you usually use but for a higher efficiency you can add SOC medium which is richer and will increase the transformation efficiency so I will add one milliliter to both tubes okay and now I will take them with them at 37 degrees with shaking to help them recover and put them into an incubator and now we wait for one hour one hour is over and I can now take myself out of the incubator and plate them so I will
have one plate for our RFP sample and one for the negative control I prefer to play at sterile environment and I will plate 100 microliters of cells for transformation so here's the negative control and the RFP sample now we'll put some beats on the plates and take them to distribute the cells so now it's well distributed I can throw away the beads and I can put the plates at 37 degrees to let them incubate overnight and hopefully tomorrow we'll see some colonies the next day I will look if there are some colonies and yes you
can see that there are some colonies on the plates with our sample and no colonies on the negative control which means that our transformation worked and we like ate it successfully the rfp into our vector and the negative control with only the vector did not give any colonies so this was the ligation and transformation protocol until next time stay positive [Music] [Music]
