Primer Designing (Manually) by ClustalW & Oligocalculator | Lecture 4 Part 2 by Dr. Muhammad Naveed
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- Опубликовано: 11 сен 2024
- Manual Primer Designing with ClustalW & Oligocalculator | Polymerase Chain Reaction is widely held as one of the most important inventions of the 20th century in molecular biology. Small amounts of the genetic material can now be amplified to be able to a identify, manipulate DNA, detect infectious organisms, including the viruses that cause AIDS, hepatitis, tuberculosis, detect genetic variations, including mutations, in human genes and numerous other tasks.
PCR involves the following three steps: Denaturation, Annealing and Extension. First, the genetic material is denatured, converting the double stranded DNA molecules to single strands. The primers are then annealed to the complementary regions of the single stranded molecules. In the third step, they are extended by the action of the DNA polymerase. All these steps are temperature sensitive and the common choice of temperatures is 94oC, 60oC and 70oC respectively. Good primer design is essential for successful reactions. The important design considerations described below are a key to specific amplification with high yield. The preferred values indicated are built into all our products by default.
#Primer #Manual_Primer_Designing #Oligocalculator
MashaAllah great effort. More power to you our mentor.
It's my pleasure
great lecture
As a student i request you to add another video lecture to primer designing series which may include primer designing for SNPs (tri and tetra).
Aoa sir these lecturers series are very helpful
It's my pleasure
Impressive. Now i can design a primer. Thank you sir.
Thank you Sir, lecture was very helpful and in simple language. May God bless you.
Pleasures and welcome dear
Very well done. jazakAllah Khair
Welcome
Bundle of thanks sir
You daid in your video thta the 3nd of the primer mist be with G OR
C and i see here some a and t ending site
Very Informative. Kindly make a video regarding degenerate primers to find out unreported genes in other species
sure
Is these primer designing methods are still reliable now a days
Thanks Dr Naveed Great work we are learning
good and welcome
Dear Dr. Naveed thank you for your lecture series. I tried to multiple align my sequence(bacteriophage ) with other( bacteriophage) plus/plus-strand but I couldn't find any conserve region. I am trying to develop overlapping primers for the whole genome to confirm the assembly of our bioinformatics analysis. Please guide.
do blast your required organism sequences then download top homologous then go for Multiple sequence alignment
@@Prof.Dr.MuhammadNaveed in this way , we design universal primer or specefic primer ??? plz respond
Sir primer design kr ny k liay Blast ko use krny ka or Clustal W ko use krny ka purpose kia h?????
Sir great work but I have suggestion, I think we should take complementary strand for reversed primer....Kindly make correction. Thanks
Yes, you are right but this reverse primer is the reverse complementary of what you said as both templates and primers and complement to each other so no matter how to design dear
@@Prof.Dr.MuhammadNaveed thanks clear nk8
Kia hum direct NCBI sy direct Sequence le kr Primer design nhi kr skty?
At Video Timeline point 11:44 / 14:27 you wrote primer Size 18 while it is 24 and one thing more shouldn't we take the complementary seq. of reverse primer?
@Dr. Muhammad Naveed please guide.
Thanks!
yes size should be between 18-24, and yes do make reverse complement of the reverse primer
Thankyou so much
pleasures
sir how we can design primers for RAPD PCR
very easy as for probe from primer3 as well as manually by justbio in lec 4
And why we use Clustal W for primer designing?
Sir forward and reverse primer Kae direction kia hogaiaee forward 5 to 3 hoga kia and reverse 3 to 5 but direction of synthesis of each primer is 5 to 3? Please tell me
yes 5-3 both
salam,,,, sir please clear just one point... ye reverse wala primer ham jo bilkul last sequence ha us se le sakty ha? ap ne thora uper wala copy kia?
where go maximum conserved region pick from there
@@Prof.Dr.MuhammadNaveed thanks... how we can make primer for cloning? is it same ?
A.o.A! Sir, just bio ky page py Jo primer anneal tool ha is ko explain Kar dain ya koi tutorial bta dain? Yani kyy hm is ko kasy run ya use karty hn?
sure will make a lecture on it
Dr. sb Please specify the ClustalW parameters. I could not find the conserved region on default parameters.
Set pairwise alignment at Slow/Accurate & sequence type at DNA. Use FASTA sequence and set all other parameters at default values.
Sir i've tried various time but i'm unable to make account on justbio. i have filled form, entered passowrd that was mailed but still its not working, what's the issue, do u have any idea?
just do copy password from and paste on justbio then c
Why we use Run Blast for primer designing?
Thank u so much sir
Thank you sir this video is very informative
Glad it helps you
Thanks sir ❤️
Very helpful lecture❤
Glads its helps you
Sir u delete some of the nucleotides in oligocal but didn't remove it from notepad
also delete form notepad
A.o.A.Sir!
Ap Ka personal no. Chaeay??
Mil skta Hy.
Blast results my a kr result select nhi ho rha.
Ap ny ISS lecture my 5 results select kiyy hn wo mhj sy nhi rhy
view comment reply on this query
What is manually?? Means
Sir reverse primer is from 3 to 5 direction or 5 to 3 direction?
design as it as forward primer but when done the do it reverse orientation means 3-5
One of my friend help me to make primer sequence of gene nrf2 in cattle but i didn’t know How to find size and assessment no of gene if I have gene sequence please guide me thanks
dear just follow primer lectures and when found conserved region then do check all primer conditions as mentioned in part 1 of this lecture further validate your primer by doing BLAST as in part 4
Dr. Muhammad Naveed thanks so much
sir kya primer intronic region sy hu tu kya wo thek amplified hu gy ???? i designed primer for ptpn22 gene having snp 1217414. kindly reply asap
yes can amplify
Sir, as you have said that in order to fix the parameter we typically delete the sequences from 3'' end i was wanting to know can we delete the repeatative sequences from somewhere inside the chosen one to get the desire Tm and GC content?
no.. we never dellete the sequence from inside. we can only delete from 3" or 5"
if find repetitive sequence (GGGGG) in centre of the primer sequence then don't select this sequence for primer designing
@@Prof.Dr.MuhammadNaveed thank you sir
Then ok
Assalamualaikum Sir!
If we select forward and reverse primers not from the start and end of the gene respectively but as you have selected then the whole gene will be amplified or the sequence between them only...?
only that area will be amplified which is in between these two primers
Shukria sir
welcome
To much long video. Can't understand.. And kindly thora easy method or ak ak kr k specify key plz
sir reverse sequence clastal w pe kuch aur haejo ap ny copy kiya wo lengthy hy thora
no only pick or download sequences from Blast which have plus/plus strand because when minus strand come on clustalW it give near to zeor homology with others
Amazing
Thank you! Cheers!
If we are designing primer for particular mutation in a exon, and the size of exon is too big so how we can design it ....so that we can amplify the pcr
The try to design set of 2 or 3 primers and after sequencing analyzed it
dear sir , kindly confirm thar its universal primer or specific primer
specific
@@Prof.Dr.MuhammadNaveed THANKS A LOT FOR UR RESPONSE SIR . jazakAllah
sir, I am working with a gene which does not contain any stop codon. If the entire sequence is coding sequence then how and from where we can pick primer?
Please do share the tutorial on primer designing for arms PCR using prime 3.
yes, might b possible if gene from an operon then stop codon may b in next gene but you may search gene annotation
How do you choose reverse primer sir?
from end of your sequence
Sir, if the primer sequence is not complementary then how would it bind to the sequence.
then it got primer and no PCR result
Good 🌷
Thank you! Cheers!
very informative and helpful thankoo how can i register my account at justbio
just click on signup/register and its take 2 minutes to add data and to be registered
you talk so fast
He talks so fast smjh nhi ati