I forgot to mention an extremely important step. So after your PCR, make sure to digest your product with DpnI for at least 1 hour at 37C. This enzyme will recognize the heavily methylated original template (non-mutated) and digest it into many fragments, and thus you will have virtually no background after your transform. If you don't digest, all your clones will be false positives!! Sorry that I forgot to say this in the video!!
It's QuikChange. Normally reverse and forward primers would not be at the same position like that. However, remember we are going to be mutating a single position on the template, which is a circular piece of DNA. The only way to accomplish this (using QuikChange anyway) is to design primers at the identical position. Each primer will go in opposite directions starting from the same position because the mutation is located there. We need to introduce mutant primers at the same place.
(cont. from previous message) Each mutant primer will introduce the mutation to both strands of DNA. Take a look starting at 30s into the video; the protocol outline explains it pretty well. Let me know if you have any other questions.
Hi You are using the primers complement to each other with mutation at the center. Will that cause over lapping at those region of plasmid after dpn I digestion and transformation into host? Also how about using reverse primer adjacent to the forward primer? Consider the sequence: Here starts forward primer CTG AGT GAT GGC GCG AAC TCG ATG GAA Here reverse primer with mutation either in forward or reverse primer at center?
Thank you so much everyone! I will be back soon and will start uploading videos once again. To Ka Yee Wong, mutagenic primers (or primers in general) usually do not need to be 40nt long. The longer they are the more difficult for them to anneal and amplify (based on my experience of using special primers over 70nt long). 20-24nt should be sufficient, although I've used ones over 30nt and they worked just fine.
I found this information very useful and is super interesting since I am new in the field. Thank you so much that you took the time to clearly explain everything. I do however, have a quick question: about the primers that you have, they can base pair complementary to one another, wouldn't that lower the yield of your mutated PCR products? I couldn't imagine. Could you help me out? Thank you so much again.
Has anybody tried the protocol described in Liu 2008; "An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol"?
hi im a little bit confused about how you made the primers. shouldn't the primer sequences be complementary to the template DNA. It look like you copied and paste the original DNA strand for the forward and backward primers?????
Hello! As you know there is also an online tool in the company website that designs primers for site-directed mutagenesis. I tried this tool and the mutations is not located in the middle of the output primers!! In your experience, which one works better? Your manual protocol or the online tool? How important is it to put the mutation in the middle of the primers?
how does the nick get fixed. you say it's done by microbial machinery but pcr is not done in vivo what is the difference between quikchange and phusion methodology
The nick becomes fixed after transformation into bacteria. If you want to know all the details comparing QuickChange and Phusion, I recommend looking up their product sheets on their websites.
Maysa Barbosa That Tm is based on the user manual. In my experience try to keep your primer Tms in the range of 60C-70C. The conditions for PCR mutagenesis is relatively flexible.
+Natalia West No, since in this example I'm using primers for mutagenesis. The only scenario in which stop codons must be included (with respect to mutagenesis) in the primer sequence would be if you're mutating (deletion, point mutation, insertion) near the stop codon in the gene sequence within the plasmid.
+Natalia West Yeah if you're doing mutagenesis it's completely dependent on the sequence you're changing. If the sequence you're changing is not near the start codon there is no need to have that in your primer sequence.
Thank you for the video, it gives me clearance. By the way, I'm a beginner in biology molecular, DNA and stuff, and it gets me a little bit frustrated to decide where should I begin to study. I study abroad now, and my Sensei told me to do the quick change for deletion, I get the general concept, but all things about sequence, region and other stuff still not so familiar for me and In here, people don't talk English so much, so it's hard for me to understand their explanation. Do you have any suggestion where can I learn basic for this material? And btw, do you know where can I get that software you use to see the sequence? Thank you so much for your help!!
Thank you for commenting. Check with the commercial product - they usually have protocols laid out and well written. The best way to learn something is just to do it. If it doesn't work, then go back and try to figure it out. From my experience, just trying the experiment will teach you many things regardless if it works or not. The software is Lasergene - it is licensed from the university I study in.
(cont. from previous message) And no, I did not copy the exact sequences. My forward and reverse primers are complements of each other at the position I want to introduce the mutation.
I forgot to mention an extremely important step.
So after your PCR, make sure to digest your product with DpnI for at least 1 hour at 37C. This enzyme will recognize the heavily methylated original template (non-mutated) and digest it into many fragments, and thus you will have virtually no background after your transform. If you don't digest, all your clones will be false positives!! Sorry that I forgot to say this in the video!!
It's QuikChange. Normally reverse and forward primers would not be at the same position like that. However, remember we are going to be mutating a single position on the template, which is a circular piece of DNA. The only way to accomplish this (using QuikChange anyway) is to design primers at the identical position. Each primer will go in opposite directions starting from the same position because the mutation is located there. We need to introduce mutant primers at the same place.
(cont. from previous message)
Each mutant primer will introduce the mutation to both strands of DNA. Take a look starting at 30s into the video; the protocol outline explains it pretty well. Let me know if you have any other questions.
With these two primers, is there any possibilities that the primer-primer dimerization would occur?
Hi
You are using the primers complement to each other with mutation at the center.
Will that cause over lapping at those region of plasmid after dpn I digestion and transformation into host?
Also how about using reverse primer adjacent to the forward primer?
Consider the sequence:
Here starts forward primer
CTG AGT GAT GGC GCG AAC TCG ATG GAA
Here reverse primer
with mutation either in forward or reverse primer at center?
Thank you so much everyone! I will be back soon and will start uploading videos once again.
To Ka Yee Wong, mutagenic primers (or primers in general) usually do not need to be 40nt long. The longer they are the more difficult for them to anneal and amplify (based on my experience of using special primers over 70nt long). 20-24nt should be sufficient, although I've used ones over 30nt and they worked just fine.
That was very helpful thank you. I was wondering what that computer program was that you were using to design the primers is?
I dont know either how those primers would hybridize with the template dna since they have the same sequence.
I found this information very useful and is super interesting since I am new in the field. Thank you so much that you took the time to clearly explain everything. I do however, have a quick question: about the primers that you have, they can base pair complementary to one another, wouldn't that lower the yield of your mutated PCR products? I couldn't imagine. Could you help me out? Thank you so much again.
Has anybody tried the protocol described in Liu 2008; "An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol"?
hi
im a little bit confused about how you made the primers. shouldn't the primer sequences be complementary to the template DNA.
It look like you copied and paste the original DNA strand for the forward and backward primers?????
Hello! As you know there is also an online tool in the company website that designs primers for site-directed mutagenesis. I tried this tool and the mutations is not located in the middle of the output primers!! In your experience, which one works better? Your manual protocol or the online tool? How important is it to put the mutation in the middle of the primers?
Hi, may i ask which DNA viewer do you use there?
how does the nick get fixed. you say it's done by microbial machinery but pcr is not done in vivo
what is the difference between quikchange and phusion methodology
The nick becomes fixed after transformation into bacteria. If you want to know all the details comparing QuickChange and Phusion, I recommend looking up their product sheets on their websites.
Thank you for the video! But, why the Tm should be as high (Tm> 78 ° C)?
Maysa Barbosa That Tm is based on the user manual. In my experience try to keep your primer Tms in the range of 60C-70C. The conditions for PCR mutagenesis is relatively flexible.
Man, you're a lifesaver!
Hey, and what happens next?
Hi, which DNA analysis program were you using?
Hi! Would you add start codons to these primers? What about using stop codons for deletions?
+Natalia West No, since in this example I'm using primers for mutagenesis. The only scenario in which stop codons must be included (with respect to mutagenesis) in the primer sequence would be if you're mutating (deletion, point mutation, insertion) near the stop codon in the gene sequence within the plasmid.
+TheScyther88 Thanks! What about start codons? During cloning, they are required in the primers - this is not the case for Quikchange?
+Natalia West Yeah if you're doing mutagenesis it's completely dependent on the sequence you're changing. If the sequence you're changing is not near the start codon there is no need to have that in your primer sequence.
Thank you for the video, it gives me clearance. By the way, I'm a beginner in biology molecular, DNA and stuff, and it gets me a little bit frustrated to decide where should I begin to study. I study abroad now, and my Sensei told me to do the quick change for deletion, I get the general concept, but all things about sequence, region and other stuff still not so familiar for me and In here, people don't talk English so much, so it's hard for me to understand their explanation. Do you have any suggestion where can I learn basic for this material? And btw, do you know where can I get that software you use to see the sequence? Thank you so much for your help!!
Thank you for commenting. Check with the commercial product - they usually have protocols laid out and well written. The best way to learn something is just to do it. If it doesn't work, then go back and try to figure it out. From my experience, just trying the experiment will teach you many things regardless if it works or not. The software is Lasergene - it is licensed from the university I study in.
Are there any certain plasmids I need to use with this?
+Emil Malta-Müller No, I'm pretty sure this will work will all plasmids. Although if the sequence is unusually G-C rich there might be issues.
Hi! would you please tell me what is the software's name you used to design a primer
+Kincaid Lu SeqBuilder
and you not answering the question, he is not asking about the position but about the sequence of each primer
Great video, you clearly explain the protocol.
Thanks a lot for simplifying it !! much more useful than reading the protocol
very helpful video thanks a lot
This was extremely helpful, thanks :)
(cont. from previous message)
And no, I did not copy the exact sequences. My forward and reverse primers are complements of each other at the position I want to introduce the mutation.
Thank you this was needed
thank you from Colombia
+Genetica Humana You're welcome!
Thanks this helped me alot
Thank you so much for this video! Helped me a lot!!!
you are awesome
Ibrahim Omoyayi I appreciate your kind words!