This RDT video discusses the properties required for designing a good primer for PCR For more information, log on to- shomusbiology.w... Download the study materials here- shomusbiology.w...
I was studying this subject and couldn't understand why the reverse primer was the complement reverse of the leading strand. Now I got it, thanks to your awesome explanation. Thanks!!!
Fir the primer diner they only form the dimer if the three from the end were consecutive meaning from 3’ end sequence they would have to be 3 in a row no gaps to form a primer dimer
Sorry, but you're completely wrong about one of the primers not binding if the temperatures are not matched. You give the example of one primer with a Tm of 55 and another of 65. You state that if you use 55 as the annealing temperature, the Tm 65 primer won't bind at all. This is incorrect. It will bind. However, as the temperature decreases to below 65, there is an increased opportunity for non-specific binding elsewhere in the template which could lead to inefficient PCR and/or artefactual amplicons.
Its realy vvv well done.. but I have one question..please.. How to add the restriction sites?? if we are going to design primers for cloning- Gene construct..??????????????????????
Hello Shomu, thank you for your videos, I got a question, I designed a pair of primers to amplify one region of a 3200 bp gene. But after sequencing, some samples yield one section of the gene and others yield other section of the gene, do you have an explanation for this? thank you.
I was wondering about the melting temperatures for the primers? For example, if one primer has a melting temperature(Tm) of 55 and another a Tm of 65. Say I lower the temperature to 60. Wouldn't the 65 Tm primer anneal but the temperature of 60 is still too high for annealing? So if I want both primers to anneal, don't I have to lower the reaction temperature to the lower of the two melting temperatures to get BOTH primers to anneal?
Dear Shomu, Thank you for the video above, it was quite helpful. I am having trouble designing new primers for HDV genotypes' G1-G8 and G1,2,4. How d I go about doing this? I have tried using NCBI.
Hello sir I've got a query, as you've said during the lecture that primer will be used to amplify the gene of our interest, so it means that we will design the primer according to our gene sequence, so my Q. is how to the identity that gene and its sequence?
Great video, but I didn't really understand the bit about melting temperature. If you're denaturing the DNA at over 90 degrees C, but the melting temperature of the primers is 50-something to 60-something degree C, what stops the primers from being damaged? Does that make sense? Is it because the melting temperature breaks the hydrogen bonds? In which case, why does heating the reaction up again to over 90 degrees not break the bonds and damage the primers? That's the one thing that has me a bit confused. If anyone in the comments section knows, I'd love to have the answer. :)
90 degrees C is high enough for the hydrogen bonds between the complementary strands to dissolve, but not enough for the covalent bonds that hold together the bases of any strand to dissolve. That is why, at 90 degrees, the two strands are disentangled from each other but are not fragmented into smaller pieces.
I want to know if the extension temp is 72 and annealing temperature is 54,so at this high temp of 72, the primer and our DNA strand should denatured but this never happens.why??
I have a question it may be a stupid one butt sir everything else is fine, but after designing pimer, from where do you put the primer in the tube, remove it from the computer or what?
Manahil , you don't remove the primers from the computer ( I know you were joking, haha). You send the designed primer details to a company which makes primers and they will produce them and send back to you in a tube. There are many such companies, like Merck. Cheer!
@@OliverCOrji u r right but primer company will not send u liquid form of primer, they send in lyophilized (powder) form u have to make it right conc. Of liquid by following the instructions of the product sheet
