Hello Sir, Your tutorial on PCR Primer designing is awesome and you cover all single details regarding it. I searched a lot and i found your tutorial on primer designing that is easy to understand. It is requested that kindly upload a tutorial on ARMS PCR primer designing as well. I am sure your tutorial on ARMS PCR will be very helpful and beneficial for me. Kindly do me a favour sir upload the video ASAP. Thankyou for your anticipation.
Thank you for your amazing explanation about primer design. I want to ask you a thing about those primers. Where will those primers attach? Are they bind in your target gene that you highlight previosly or in other target gene sequence? And how about the orientation of reverse primer after convert into forward primer, is it still 5'-3' or change into I am willing to hear your explanation as it is important for my first experience. Thank you so much, Prof 🙏.
Dear sir, first of all thanking your informative video and i have a doubt that if we include introns region into template sequence does it make probelm when we are following Reverse transcriptase pcr with RNA extract of blood samples
thanks for this video sir, it is helped me a lot. please make a video if possible on Primer designing for Expression vectors and identification of ER sites for cloning.
hii... BB, Can you please make again one more video for PCR- Primer as well as Probe designing video in details with more time. as its been more than 2 years you uploaded this. make with latest primer-probe design tutorials with min. 1hr length so that we can understand it properly. consider we dont know anything from scrap to full completion. Can you make it, its very humble request. because its very impactfull technique & you know very better how to use it. there are many videos like it. dont contains all. i hope if you will be making it will be best of them all. thanks.
Nhiệt độ tan chảy (melting temperature - Tm) tối ưu của các đoạn mồi chạy PCR là bao nhiêu? Làm sao tăng hay giảm Tm? Ngoài chỉ số Tm thì khi chọn đoạn mồi chúng ta cần phải chú ý tới thông tin nào? Và ý nghĩa của thông tin đó là gì?
Thank you professor, I want linearized vector it has 7000 basepair. I tried restrictions enzyme, but I can't find valid enzyme.so I want to make primer. Please guide me. I also tried to make primers but I can't fullfil the condition of tm, gc and no of basepair. How can I do.
Thank you for the informative video. I want to design primer(s) for COI (1533 bp). I don't want to amplify pseudogene (numts). In the video, you mentioned that if the sequence is more than 1000, the target region should be divided into two. Divided into 2 halves?.
Excuse, i want to ask if i am not working on gene cloning; rather, I am conducting gene expression experiments. How can I avoid incomplete gene amplification when designing primers?
Thanks for the awesome video! Keep up the good work. I do have a question for clarification. For example you have picked your forward and reverse primers, all passed the qualities. Then you BLAST your forward and reverse primer sequences individually to check if the primers will capture different sequence aside from your target molecular marker. Example: F and R primers are intended to amplify ssuRNA of a pathogen but when you blast your primer sequence it shows that has a hit to different organisms like COI of plants. What is your judgement on this case? I hope i explained it clearly and looking forward to hear from you. Thanks!
Your welcome... It's quite common , some time your sequence will match with other organism, here you have to understand evolution concepts. In evaluation some sequence region are conserved throughout the species, for this you need not worry , why means your looking for a sequence in specific organism not in multiple organism.. what u need to worry is if ur primer sequences are match with the same organism of different gene regions, then u have to redesign the primers
@@Bioinformaticswithbb thank you for your insights Sir. I will take note of this. I have another question if you dont mind. Is it advisable to perform multiple sequence alignment of your molecular marker from different species of the same genus? For pathogen detection, there are virulent genes shared by different species of pathogen under one genus. Its just that sometimes it varies/strains. I have read that finding the conserve region of sequences is recommend to design specific primers for that pathogen.
I didn't it from 10:34, range for forward and reverse primer. Why did not you mention target region in the range of forward primer? Is not your goal to amplify the target regions?
Hello sir, I have some question. pcr primer is the complementary sequence of dna strand of the target region where it is bind. But forward primer in this tutorial not complementary. It is the dna sequence of intron. I don't understand about it. please if you clarify this it will helpful. Another one is, the forward primer is far from the target region. When taq polymerase start to copy, unwanted part also copy along with target sequence. Is it not create any problem? Thank you.
Once u upload ur sequence primer design tool, it will develop set of primers for the sequence i.e., Forward and Reverse. Forward mean it direction is 5`>3` Reverse mean it direction is 3`>5` (U don’t find this sequence in above example video, why mean its in reverse complement) Why we design to set of primer?, As u know we have double stranded DNA each stand is complimentary to other, our aim with pcr is we need to synthesize our interested DNA region in both stands, for this reason we use reverse and forward primers. if u have further clarification u can join our our facebook group and past ur comments. facebook.com/groups/261045198486665/
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Very well explained, covering both primer design and validation! Worth viewing for beginners!
Very well explained sir. Congratulations and expecting many more for genetics community.
Thanks, Prof. This is the most comprehensive video on primer design I have ever watched.
Well explained. Thank you for sharing
my pleasure to call you real a prof. of Abstract solver in Primer design.
Your always welcome
Hi sir I'm biggest fan for your teaching. You are just outstanding teacher
very informative lecture , thank for sharing your knowledge . plz give knowledge of ARMS and RFLP PCR designing from zero .
Hello Sir, Your tutorial on PCR Primer designing is awesome and you cover all single details regarding it. I searched a lot and i found your tutorial on primer designing that is easy to understand. It is requested that kindly upload a tutorial on ARMS PCR primer designing as well. I am sure your tutorial on ARMS PCR will be very helpful and beneficial for me. Kindly do me a favour sir upload the video ASAP. Thankyou for your anticipation.
You helped me complete my assignment, Thank you so much!
perfectly explained
❤❤❤❤❤
This video was really very helpful for beginner's. Thanks a lot for sharing everything in detail and hoping to see more tutorials like this.
What a useful video thank you !
Great explanation
Thanks for the refresher. It was certainly worthwhile. Thanks so very much.
Glad you enjoyed it!
You literallyyyy saveeeeeed me ❣️
Thank you so much Prof BB, this was super helpful!
Tysm sir😊
Thank you for your amazing explanation about primer design. I want to ask you a thing about those primers. Where will those primers attach? Are they bind in your target gene that you highlight previosly or in other target gene sequence? And how about the orientation of reverse primer after convert into forward primer, is it still 5'-3' or change into
I am willing to hear your explanation as it is important for my first experience. Thank you so much, Prof 🙏.
Nice
Thanks
Dear sir, first of all thanking your informative video and i have a doubt that if we include introns region into template sequence does it make probelm when we are following Reverse transcriptase pcr with RNA extract of blood samples
thanks for this video sir, it is helped me a lot. please make a video if possible on Primer designing for Expression vectors and identification of ER sites for cloning.
Sir, what if we want to introduce a restriction site in the primer for cloning. What should we do then?
Very informative
Excellent....best wishes
..
Rabbani,
Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka, Bangladesh
Thank you so much for this information sir...it helped me alot
well explained sir. Sir i have a doubt on cds, exon, gene selecting options. What we have to choose if we are making qRT pcr .
hii... BB, Can you please make again one more video for PCR- Primer as well as Probe designing video in details with more time. as its been more than 2 years you uploaded this. make with latest primer-probe design tutorials with min. 1hr length so that we can understand it properly. consider we dont know anything from scrap to full completion. Can you make it, its very humble request. because its very impactfull technique & you know very better how to use it. there are many videos like it. dont contains all. i hope if you will be making it will be best of them all.
thanks.
Thank you sir.
really great ,,it will be appreciating if you will describe vector designing also
Nhiệt độ tan chảy (melting temperature - Tm) tối ưu của các đoạn mồi chạy PCR là bao nhiêu? Làm sao tăng hay giảm Tm?
Ngoài chỉ số Tm thì khi chọn đoạn mồi chúng ta cần phải chú ý tới thông tin nào? Và ý nghĩa của thông tin đó là gì?
Hello sir can you make video on how to design siRNA
Thank you professor, I want linearized vector it has 7000 basepair. I tried restrictions enzyme, but I can't find valid enzyme.so I want to make primer. Please guide me. I also tried to make primers but I can't fullfil the condition of tm, gc and no of basepair. How can I do.
thanks
Thankyou so much sir
Why to select 100bp away from the target region?
Thank you for the informative video. I want to design primer(s) for COI (1533 bp). I don't want to amplify pseudogene (numts). In the video, you mentioned that if the sequence is more than 1000, the target region should be divided into two. Divided into 2 halves?.
Very helpful, Thanx
Thank you, sir.
Ur welcome...
Awesome description. But I want to know one thing. How can we check the primer hits? Whether the selected primers give single or multiple hits.
Run in silico pcr for the primer to test its efficiency before ordering
Excuse, i want to ask if i am not working on gene cloning; rather, I am conducting gene expression experiments. How can I avoid incomplete gene amplification when designing primers?
What if there is no RefSeq gene?
Thankyou sir
Can we take 16 nucleotide for primer designing ?
i couldn't find the highlight sequence feature
Me tooo
How do I elongate the RNA sequence?
Thanks for the awesome video! Keep up the good work. I do have a question for clarification. For example you have picked your forward and reverse primers, all passed the qualities. Then you BLAST your forward and reverse primer sequences individually to check if the primers will capture different sequence aside from your target molecular marker. Example: F and R primers are intended to amplify ssuRNA of a pathogen but when you blast your primer sequence it shows that has a hit to different organisms like COI of plants.
What is your judgement on this case? I hope i explained it clearly and looking forward to hear from you. Thanks!
Your welcome... It's quite common , some time your sequence will match with other organism, here you have to understand evolution concepts. In evaluation some sequence region are conserved throughout the species, for this you need not worry , why means your looking for a sequence in specific organism not in multiple organism..
what u need to worry is if ur primer sequences are match with the same organism of different gene regions, then u have to redesign the primers
@@Bioinformaticswithbb thank you for your insights Sir. I will take note of this. I have another question if you dont mind. Is it advisable to perform multiple sequence alignment of your molecular marker from different species of the same genus? For pathogen detection, there are virulent genes shared by different species of pathogen under one genus. Its just that sometimes it varies/strains. I have read that finding the conserve region of sequences is recommend to design specific primers for that pathogen.
I didn't it from 10:34, range for forward and reverse primer. Why did not you mention target region in the range of forward primer? Is not your goal to amplify the target regions?
Please I have humain gene (hla) how i do primer design
I have the protocols for normal PCR.how can I optimize them for using multiplex PCR
Steps are similar... but ur selection of the sequence is the ky in multiplex PCR .. refer this premierbiosoft.com/tech_notes/multiplex-pcr.html
How to decide target site
It works also for rt qPCR?
Where is the primer specifity check???? your primers have other non-specific targets
Sir, if my gene of interest is having 50 exons. So I Need to prepare primers for all exons? I just wanted to check either the gene expressed or not.
No need .. if want to cover 50 exons advisable to go for whole Exome sequencing
Good morning sir, I am Manohar Naik your Junior in sku from Kadiri sir , I have a doubt how can I contact you sir I have no number sir
Hello sir,
I have some question. pcr primer is the complementary sequence of dna strand of the target region where it is bind. But forward primer in this tutorial not complementary. It is the dna sequence of intron. I don't understand about it. please if you clarify this it will helpful.
Another one is, the forward primer is far from the target region. When taq polymerase start to copy, unwanted part also copy along with target sequence. Is it not create any problem?
Thank you.
Once u upload ur sequence primer design tool, it will develop set of primers for the sequence i.e., Forward and Reverse.
Forward mean it direction is 5`>3`
Reverse mean it direction is 3`>5` (U don’t find this sequence in above example video, why mean its in reverse complement)
Why we design to set of primer?, As u know we have double stranded DNA each stand is complimentary to other, our aim with pcr is we need to synthesize our interested DNA region in both stands, for this reason we use reverse and forward primers.
if u have further clarification u can join our our facebook group and past ur comments.
facebook.com/groups/261045198486665/
@@Bioinformaticswithbb thank you very much i had this same confusion but now its clear.