Thanks for the very good explanation! One question. XhoI restriction site is 5’-CTCGAG-3’. However, in the reverse primer example in this video, the added XhoI RE site is written as 5’-GAGCTC-3’. Is the nucleotide sequence of the XhoI in the reverse primer correctly written? Thank you!
@@katharinehubbard5043hi there. This is by no means a criticism, as I think this video is really helpful, but I would wholeheartedly recommend amending it so the 5’-3’ reverse primer is correctly written. I lost about a month of work for this reason, and I know I should’ve read the comments beforehand, but still, I think it’s worth amending.
You are adding in the reverse primer (when you write It 3'-5') the sequence of the MCS in 3'-5' but that sequence that you added is actually the MCS written in 5'-3' (i ve searched the sequence). So, do you have to change the order of the MCS into 3'-5', dont you?
A lovely video ❤ however there's something i don't understand, in nested pcr, a primer amplifies a specific region in a sequence but i don't understand how, since a Primer is supposed to amplify from the binding site to the end no?
Sorry i can't articulate my thoughts well since English isn't my first language, but for example i have a 600 nucléotide sequence and i want to amplify a 200 region inside of it but let's say i have a primer that start at 200 isn't going to amplify everything from 200 to 600?
@zizojaezekeom3565 Good question! Initially, the polymerase will actually continue to amplify all the way until the end of the DNA strand, and you won't find any of our target/limited DNA sequences. However, after the third cycle of PCR, remember that the primer going in the opposite direction will bind to overextended single strands of DNA so that when elongation happens again, now your product is fully restricted/limited to your target sequence. See this video for an explanation and animation: ua-cam.com/video/2KoLnIwoZKU/v-deo.html
Professor Hubbard you're an outstanding instructor. I have a question: Going in the 5" direction, does the first letter of a consensus sequence go at -10, or is it the last letter? Anyone on here who knows may answer.
Hye Mam, Plz guide me about this primer specifications, if it is 30-35 bp length. So what about is Tm, GC content it increased significantly. How we control this, or we set the Tm and GC content according to Start codon part and gene of interest part in primer and excluding the add-on restriction enzyme and 6 base pair sequence
Dear Dr. Hubbard, thanks for the lucid lecture. Could you please let us know, does a stop codon derived out of reverse primer affects the protein expression.
thankss, could I write you an email for an example of primers designing? Because I think my professors were wrong.. I'm going crazy trying to understand
How I can implement this strategy to get rid of two tags; N-terminal 6x His tag and C terminal Avidin tag. flanking the gene of intrest that codes for certain protein ?
how come ATC is a stop codon? Also could you please add some more factors while designing a primer like annealing and melting temperatures, GC content etc.?
Thank you very much, a very straightforward explanation. I have a question: what happens if the designed primers have very different Tm between them. Is it possible to design the primers before or after the gene sequence to avoid this and also get better CG percentages? If not, is there another approach to increase the PCR efficiency if the gene sequence only allows to design a non compatible pair of primers?
If your primers have very different Tm this might cause issues with your PCR. Sometimes in cloning this can’t be helped, but sometimes just moving the start position by a few bases, or lengthening one primer can really help if this gives you a more similar CG percentage.
Does the strand (forward or reverse) of the gene matter when designing a primer? Let's say a gene is located in the forward strand; should we design forward and reward primers the same way as for a gene located in the reverse strand?
Very well explained. Saved so much of my time going through textbooks/papers. I have one question. In PCR, the template strand will melt and then the primer will bind to the complementary sequence of the template. So won't the primer sequence be (if started from the start codon for eg) 3'-TACGTTGCCATGGC-5'? which will then go and attach to the template and the polymerase will go on adding nucleotides in 5' direction of the primer.
During the first cycle the artificially added bases (ie the restriction site) don't bind to the template as they are not complementary, but the rest of the primer is sufficiently complementary at the 3' end for the polymerase to bind and initiate the reaction. For all subsequent cycles the primers become the start of the strand as usual, so all amplified pieces of DNA contain the restriction site at the start. Hope that helps!
Great video, and thanks. One possibly silly question - the primers are here described as being within the amplicon, the sequence for the GOI. Aren't they supposed to be in the flanking regions ? Not saying its a mistake, I'm just confused about this issue...
Isn't the forward primer usually complementary to the sense strand and not identical with the sense strand sequence? So that the forward primer binds to the sense strand..
This is a common misconception - the forward primer becomes part of the new sense strand - the polymerase elongates from the 3’ end of the primer using the antisense strand as a template. It therefore is the identical sequence to the sense strand
tysm for this, i have a whole cloning hw and im trying to figure out how to design primers. one question, do i have to start my fwd primer from the start codon and end my rv primer at the stop codon? thanks sm for the answer in advance
It depends what you want to amplify! If you want to amplify the open reading frame (protein coding region) then you need to have primers that include the stop and start codon. But you might also want to amplify the promoter or the 5’/3’Untranslated regions (UTRs). Or if you want to add a tag or fusion protein you might want to stop before the stop codon, assuming there is a stop codon at the end of your fusion. Really depends on your requirements! Hope that helps :)
This video sums up all the essential aspects but i was wondering just one thing that during simple pcr we amplify the sequence by foward primer attached to the bottom strand while for reverse primer we use the upper strand. So it's not the same case here since we are using pcr capture? Can you explain it a bit?
6:50 wouldnyt you have loose ends as the PCR product bc the artificial site isn't actually apart of the backbone, so the elongation will only go as far as the black bit?
In the first cycle you are right there would be an overhang as the artificial part of the primer has nothing to anneal to. In subsequent cycles the polymerase will extend the full length of the primer (it can’t tell the difference between primer and original DNA in the template strand) so the final PCR products will extend to include the primer region on both strands with a blunt end. I could have been clearer about this taking multiple rounds of replication in the video. Hope that helps
very informative video and explained very well. How do we add protein tag at 5` terminal of forward primer? Do we need to put atg before the tag and then our goi sequence?
@@katharinehubbard5043 the gene is starting from atg. So it would be atg then tag then the gene sequence starting from atg... right? like this ATG-Tag sequence-ATGTGGC like this right?
@@ManitaRaina Yes - translation will start from the first ATG (AUG in the mRNA) and then everything will be translated from then on. ATG-TAG-GeneSequence. You could technically remove the second ATG at the start of the gene sequence and it would still work as long as you've got one in before the tag. Hope that helps!
very helpful video! I have a doubt..is it necessary to change the stop codon in reverse primer to AAA..won't it affect primer binding or won't it change the insert sequence after amplification..if I need to subclone the insert into another vector?
You can change the stop codon to any other codon - I used AAA as an example. It will reduce the binding affinity of the primer, hence why you need a decent length region that is able to bind to the sequence within your primer. It will change the insert sequence from stop to the relevant codon - that's the point! You only replace the stop if you want to create a longer fusion protein or add a tag - if you just want to sub clone the gene you would keep the stop codon in your primer. Hope that helps!
After removal of the stop codon, does the tag go upstream of the restriction site? Also, do we add a new stop codon farther downstream of the original one?
Why not just remove the stop codon all together instead of replacing it with a random codon? GoI->Restriction site sequence without anything in between
Thank you so much for making this! I've spent hours trying to understand how to do this and you cleared up all my confusion in just 20 minutes.
I have an exam in 3 hours and this just saved my grade. Thank you Katharine!
3 weeks of my lab explained In 20 min.....Thank you so much for this resources
Im a Student in Germany.. your video ist more than excelent.. thank you a lot ..
Thank you so much! Your explanation was very thoughtful and straightforward-I appreciate it!!
thank you so much！Your explanation about the stop codon really helps me a lot with my assignment.❤
thanks mam, you save my money and time. God bless you. No one had explain the STOP CODON properly
Truly well done, thank you for your insight!
Thank you for sharing! Very informative! You're such an effective educator!
You are exceptional, plz also make videos on homologous recombination, expression plasmid and chromosomal integration too
Thank you for this great video! Very informative!
you cleared up all my confusion in just 20 minutes
Prof Hubbard, thanks a lot for your excellent video. very well explained.
That was a great video! Simple and very well explained. Thanks a lot!
AMAZING.. WONDERFUL CLEAR EXPLANATION
Incredible video thank you so much !!!
Your videos are amazing! Thank you
Thanks for the very good explanation! One question. XhoI restriction site is 5’-CTCGAG-3’. However, in the reverse primer example in this video, the added XhoI RE site is written as 5’-GAGCTC-3’. Is the nucleotide sequence of the XhoI in the reverse primer correctly written? Thank you!
Well spotted and apologies for the error - you are absolutely right that it is written in the wrong orientation, so should be 5’-CTCGAG-3’.
@@katharinehubbard5043 Thank you very much!
@@katharinehubbard5043hi there. This is by no means a criticism, as I think this video is really helpful, but I would wholeheartedly recommend amending it so the 5’-3’ reverse primer is correctly written. I lost about a month of work for this reason, and I know I should’ve read the comments beforehand, but still, I think it’s worth amending.
you are incredible !! Thank you.
Found this useful. Thank you
Amazing video, thank you!
These videos are excellent. Thanks so much 👍
Really a simple and good explanation. Great job 👍
Thank you for the video. Very informative
WHAT A GREAT DOCOTR !!!
The best explanation!
Loved this, thanks!
Excelent video, thank you so much!!
Thanks from Nigeria...
Thank you so much ma'am, this video really helped me a lot :)
You are adding in the reverse primer (when you write It 3'-5') the sequence of the MCS in 3'-5' but that sequence that you added is actually the MCS written in 5'-3' (i ve searched the sequence). So, do you have to change the order of the MCS into 3'-5', dont you?
Great detailed explanation!
Thanks you very much very very very much handy!
Great Video!
A lovely video ❤ however there's something i don't understand, in nested pcr, a primer amplifies a specific region in a sequence but i don't understand how, since a Primer is supposed to amplify from the binding site to the end no?
Sorry i can't articulate my thoughts well since English isn't my first language, but for example i have a 600 nucléotide sequence and i want to amplify a 200 region inside of it but let's say i have a primer that start at 200 isn't going to amplify everything from 200 to 600?
@zizojaezekeom3565 Good question! Initially, the polymerase will actually continue to amplify all the way until the end of the DNA strand, and you won't find any of our target/limited DNA sequences. However, after the third cycle of PCR, remember that the primer going in the opposite direction will bind to overextended single strands of DNA so that when elongation happens again, now your product is fully restricted/limited to your target sequence. See this video for an explanation and animation: ua-cam.com/video/2KoLnIwoZKU/v-deo.html
what is the purpose of adding additional nucleotides at the end ?
DAMET garm , merci TOPOLI
how to design primers if we want to add a tag to protein?
Professor Hubbard you're an outstanding instructor. I have a question: Going in the 5" direction, does the first letter of a consensus sequence go at -10, or is it the last letter? Anyone on here who knows may answer.
Please can you tell the tool to design this type of primers?
You are brilliant thanks a lot
Hye Mam,
Plz guide me about this primer specifications, if it is 30-35 bp length. So what about is Tm, GC content it increased significantly. How we control this, or we set the Tm and GC content according to Start codon part and gene of interest part in primer and excluding the add-on restriction enzyme and 6 base pair sequence
Dear Dr. Hubbard, thanks for the lucid lecture. Could you please let us know, does a stop codon derived out of reverse primer affects the protein expression.
thankss, could I write you an email for an example of primers designing? Because I think my professors were wrong.. I'm going crazy trying to understand
Great job 👍
How I can implement this strategy to get rid of two tags; N-terminal 6x His tag and C terminal Avidin tag. flanking the gene of intrest that codes for certain protein ?
how come ATC is a stop codon?
Also could you please add some more factors while designing a primer like annealing and melting temperatures, GC content etc.?
Also tell me mam, it is necessary to add these 6 bp sequence, we reduce its number or not. Also tell me it cause primary structure formation or not
Thank you very much, a very straightforward explanation. I have a question: what happens if the designed primers have very different Tm between them. Is it possible to design the primers before or after the gene sequence to avoid this and also get better CG percentages? If not, is there another approach to increase the PCR efficiency if the gene sequence only allows to design a non compatible pair of primers?
If your primers have very different Tm this might cause issues with your PCR. Sometimes in cloning this can’t be helped, but sometimes just moving the start position by a few bases, or lengthening one primer can really help if this gives you a more similar CG percentage.
I Lov it
don't the two primers add to different stands of the target dna? then how did both the primers end up in the same dna in the second step in the pcr?
extremely useful!
Great video...
but, I have a query
Xho1 is 5 CTCGAGG 3
So the REVERSE sequence should be as follows as I guess
5 xxxxCTCGAGCTATTTGGCGGC 3
You are correct - there is another comment from @separase below who spots the same mistake. Apologies for any confusion caused!
How do you know the restriction codon?
If someone at your laboratory deliberately left 5 mL of T7 exonuclease gene 6 out at room temperature for one hour what would said to them?
Does the strand (forward or reverse) of the gene matter when designing a primer? Let's say a gene is located in the forward strand; should we design forward and reward primers the same way as for a gene located in the reverse strand?
Very well explained. Saved so much of my time going through textbooks/papers. I have one question. In PCR, the template strand will melt and then the primer will bind to the complementary sequence of the template. So won't the primer sequence be (if started from the start codon for eg) 3'-TACGTTGCCATGGC-5'? which will then go and attach to the template and the polymerase will go on adding nucleotides in 5' direction of the primer.
Great video Dr.Hubbard ! A question: how does the (R. Sites added in blue) anneal to your vector backbone (dashed grey color ) during PCR
During the first cycle the artificially added bases (ie the restriction site) don't bind to the template as they are not complementary, but the rest of the primer is sufficiently complementary at the 3' end for the polymerase to bind and initiate the reaction. For all subsequent cycles the primers become the start of the strand as usual, so all amplified pieces of DNA contain the restriction site at the start. Hope that helps!
@@katharinehubbard5043 Thank you Dr! hope you will post other helpful videos
Great video, and thanks. One possibly silly question - the primers are here described as being within the amplicon, the sequence for the GOI. Aren't they supposed to be in the flanking regions ? Not saying its a mistake, I'm just confused about this issue...
Isn't the forward primer usually complementary to the sense strand and not identical with the sense strand sequence? So that the forward primer binds to the sense strand..
This is a common misconception - the forward primer becomes part of the new sense strand - the polymerase elongates from the 3’ end of the primer using the antisense strand as a template. It therefore is the identical sequence to the sense strand
@@katharinehubbard5043 Thank you for the clarification! :)
tysm for this, i have a whole cloning hw and im trying to figure out how to design primers. one question, do i have to start my fwd primer from the start codon and end my rv primer at the stop codon? thanks sm for the answer in advance
It depends what you want to amplify! If you want to amplify the open reading frame (protein coding region) then you need to have primers that include the stop and start codon. But you might also want to amplify the promoter or the 5’/3’Untranslated regions (UTRs). Or if you want to add a tag or fusion protein you might want to stop before the stop codon, assuming there is a stop codon at the end of your fusion. Really depends on your requirements! Hope that helps :)
Hi, the reverse primer should be 5-CTCGAG or 5-GAGCTC?
I am confused now😅
can you please explain why is primer not complementary
This video sums up all the essential aspects but i was wondering just one thing that during simple pcr we amplify the sequence by foward primer attached to the bottom strand while for reverse primer we use the upper strand.
So it's not the same case here since we are using pcr capture?
Can you explain it a bit?
The videos I watched followed the convention of:
Upper:5' to 3'(reverse primer)
And bottom:3' to 5'(forward primer)
thank you
Very nice lecture. Thank you. How to determine lead sequence before restriction sites ?
The sequence before the restriction sites can be anything - after digestion it will be removed
6:50 wouldnyt you have loose ends as the PCR product bc the artificial site isn't actually apart of the backbone, so the elongation will only go as far as the black bit?
In the first cycle you are right there would be an overhang as the artificial part of the primer has nothing to anneal to. In subsequent cycles the polymerase will extend the full length of the primer (it can’t tell the difference between primer and original DNA in the template strand) so the final PCR products will extend to include the primer region on both strands with a blunt end. I could have been clearer about this taking multiple rounds of replication in the video. Hope that helps
@@katharinehubbard5043 that makes more sense to me now, thanks!
If I was to add in a hexa his-tag at the N-terminus with a cleavage site, do I add that before or after the gene of interest sequence ?
If you want to add at the N terminus you need to put the tag before your gene of interest i.e. at the 5'end
@@katharinehubbard5043 ah okay thank you very much, your video made the topic really easy to grasp aswell as the other ones
very informative video and explained very well. How do we add protein tag at 5` terminal of forward primer? Do we need to put atg before the tag and then our goi sequence?
Yes - you need ATG as your start codon, then your tag, then your gene of interest. This will give the tag at the N terminus of the protein.
@@katharinehubbard5043 the gene is starting from atg. So it would be atg then tag then the gene sequence starting from atg... right? like this ATG-Tag sequence-ATGTGGC like this right?
@@ManitaRaina Yes - translation will start from the first ATG (AUG in the mRNA) and then everything will be translated from then on. ATG-TAG-GeneSequence. You could technically remove the second ATG at the start of the gene sequence and it would still work as long as you've got one in before the tag. Hope that helps!
@@katharinehubbard5043 Thankyou so much for the insight. Been very helpfull🤝🤝
very helpful video!
I have a doubt..is it necessary to change the stop codon in reverse primer to AAA..won't it affect primer binding or won't it change the insert sequence after amplification..if I need to subclone the insert into another vector?
You can change the stop codon to any other codon - I used AAA as an example. It will reduce the binding affinity of the primer, hence why you need a decent length region that is able to bind to the sequence within your primer. It will change the insert sequence from stop to the relevant codon - that's the point! You only replace the stop if you want to create a longer fusion protein or add a tag - if you just want to sub clone the gene you would keep the stop codon in your primer. Hope that helps!
@@katharinehubbard5043 Okk, I understood now. Thank you so much!
After removal of the stop codon, does the tag go upstream of the restriction site? Also, do we add a new stop codon farther downstream of the original one?
Take a look at my protein tags video which should answer your questions! Plasmid design - protein tags
ua-cam.com/video/GlkanPmCzKw/v-deo.html
you're a queen
very well explained Katharine , but when we design for a reverse primer the reverse complement of the gene of interest need to be taken
You made a mistake, restriction site sequence on the reverse primer should be the other way round, you included the 5'-3' sequence in a 3'-5' sequence
Yes I know - a few other people have spotted it in the comments below. Will try and edit to make the mistake more obvious in the video!
:)
Hello, Would you be interested in doing private tutoring online? Do you have an email I could contact? Thank you
You did not design the reverse primer correctly... It should be reverse complementary, not simply a reverse nucleotide sequence.
Why not just remove the stop codon all together instead of replacing it with a random codon? GoI->Restriction site sequence without anything in between
That also works!
Thank you so much for making this! I've spent hours trying to understand how to do this and you cleared up all my confusion in just 20 minutes.