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!!!
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.
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
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?
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..??????????????????????
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??
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?
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.
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 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
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!!!
You saved me as I am studying for the final examination... thank you so much
You're welcome. Glad to hear that you're getting benefit from my lectures
thank you so much. a 4 hour lecture in class did a horrible job of explaining what you explained so easily in 20 minutes. thank you
Viewing from Kenya, with an example coming; you are really a saviour Sir.
Thank you so much for appreciating my efforts
Your explanation is better than my lecturer !!! Thank you so much, u save my final paper tmr !!!
Thank you so much for appreciating my efforts
Thank you sir i always see you lectures when cannot under stand the topics .thuk you so much.
may God bless you with happiness and success in life.
Glad to hear that you're getting benefit from my lectures
The way you explain this things Shomu. So helpful
Thank you so much for appreciating my efforts. Glad to hear that you're getting benefit from my lectures
U saved my Final paper man... Thanx
You're welcome. Glad to hear that you're getting benefit from my lectures
nicely explained. Best teacher for Biology and biotechnology
Thank you so much for appreciating my efforts. Glad to hear that you're getting benefit from my lectures
Thanks Shomu! You're basically saving me on accomplishing my master degree hahahaha!
Thank you for your simplest explanations ❤
You're welcome
Thank you sir I got it a lot of knowledge from your video actually I work in molecular laboratory
Thank you so much for appreciating my efforts
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.
Tm of primer shud nt be more thn 5,as u said non sp.binding or incorrect bndng occur,true
Amazing brain!! So helpful...appreciate ur knowledge level
Glad to hear that you are getting benefit from the videos
@@shomusbiologyofficial i have some confusion regarding primar designing... Can I ask and get help for those ?
Decent summarization of the topic. How about a video on the proper step by step design of primer. A simple tutorial
Great Shomu. You are the best
+Vinie Kouamou thank you. Glad you liked my lectures
Best one you are for me sir ..... literally ......Thank you so much sir 😊😊
You're welcome
Very well discussed in the shortest possible time Shomu. aaro video koro. sabash.
Very thoroughly explained. Thank you for sharing💝
You're welcome. Glad to hear that you're getting benefit from my lectures
this is very good suman. it is the easiest way to explain primer design
thank you
Thanks so much sir. I love you. U simplify my work ❤️❤️❤️❤️❤️❤️❤️
You're welcome. Glad to hear that you're getting benefit from my lectures
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
Thank you very much. It helps me understand this within 30 min.
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.
Thank you very much. It's very clear. This RTD helps me a lot.
Thank you so much for this thorough explanation! It helps a lot.
Thank you very much for this informative tutorial! Congratulations on the good work!
amazing..thanks a lot for making me understand this process for the first time.
Thank you very much. Very informative and helpful. Congratulation!
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?
Jazakallah sir..., 👍💖
Form Pakistan
You're welcome. Glad to hear that you're getting benefit from my lectures
Sir can you please explain the difference between runs and repeats and secondary structure. Thankyou ✨
Okay
tq so much.. it helps me understand this within one night...
Wow ......powerful explanations.....this is really cool
Thank you sir.Very good explanation.
Very helpful, thank you so much
You're welcome
I didn't get it. Did you mean annealing temperature instead of melting temperature ?
amazing video...
my question is how to get to know these information e.g melting point of a specific primer?
There are websites that predict Tm. Try using NEBasechanger or smith like that
Love u sir 💗💗
Thank you
Can u please do video on how to select primers in NCBI and how to do blast ? What are the mistakes to avoid?
thanks brother for such explaining , you are great
This was very helpful, thank you for sharing.
Its really cool. thank you very much for helping others.
u made my life easy!!!!!! thanks a bunch!!!
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..??????????????????????
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??
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?
Good morning sir
Can you please tell me if short primer gives unwanted binding then what happened with out of range very long primers??
Thanks a lot but I am not clear with the "distance between base pairs" I just got confused as to how do we manage that?
Best explained....thanks sir
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.
Thanku so much, it is so helpfull
Glad you liked my lectures
very lucid way of pesentation
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.
Ah, that makes sense. Thanks very much for your answer.
Sir, pls clear what will happen if we have 90%GC content??
🙏
thank you soo much it was really helpful
Excellent video.
why are you so smart? :D thanks for the explanation
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
Thanks a lot ... that was well explained and brief :)
can you please make video for control of PCR contamination
I wonder what is your education level?
Great video 🤗☺👍
You're welcome
Hii, Which software you are using?
thank u very much, it's very understandable and informative
Thanks
You're welcome
Are you indian
What is gen ?
how to i download this video ?
Thank u very much.
BestOne
Thank you
Thanks for sharing !
Very informative
Thank you !
❤️❤️❤️
thank u
You're welcome
😍
Thank you
excellent
thanks alot
👍
good one
excelent
general sibhat efrem , symbol kalsi hzbi ertra eyu, ab xelim africa hzbawi gnbar kabzefreyetom zbelexe general eyu.eti ab mengo esseyasn dr. abiyn ztegebre mstirawi smimie ktgber entekoynu general sibhat eti zabeye enkifat kikewn kemzikiel sleztegemete eyu b esseyas bmedeb teharimu.fetene kitlet sibhat efrem mejemeri mtfaen mdmsasn kalsi hzbi ertra eyu.bzkone engeener tgebro zeleka xaeri kexilelu, entekone ab lieli tegaru zeleka areaeya keyr eka, meknyatu abzi seat ezi eti gchit nxur eyu.beti hade wegen oromo+opressed eritreans+tegaru versus neftegna+blxigna party+HGDEF+EZEMA.slezi tegaru strategic mehazut kalsina eyom abatom atekirka tgebro gosguas ktkiyr ymhxeneka.kalie betgebro kalsi zelene adnakot kgelxelka ydeli, ajoka kexil
Thanks
You're welcome
❤️❤️❤️
Thank you