Addgene does not regularly monitor comments posted here, so we may not see your question immediately. We’d be happy to answer any questions sent to help@addgene.org as soon as possible. Please include the name of the video along with any questions so our support team can help. Thanks!
Thanks. You speculated me with a standard presentation. I have one question if you don't mind. You said to add 4 degree for each pyrimidine base and 2 degree for each purine base to calculate melting temperature of the primer. Can you explain why I should add 4 or 2 in those perticular cases?
She explained later in the video, that C-G base pairs have 3 hydrogen bonds, while A-T pairs have just 2, therefore G-C interactions are stronger than A-T. hence the higher temperature required to melt G-C compared to A-T
Thank you for your question. As long as you know the genomic sequence of the virus you are interested in, you can design a primer for that virus following the same tips and guidelines described in this video. You can also check out Sigma Aldrich’s page on PCR assay design for more information: www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/pcr-qpcr-dpcr-assay-design
Thanks for the question! An annealing temperature 5C below the Tm can be used as starting point for PCR, but this temperature is something that may need to be optimized for high yields. A temperature of 5C below the Tm should allow for annealing to the expected target site. Lowering the annealing temperature further though could increase annealing to off target sites that differ by single nucleotide mismatches.
in case of primer dimer / hairpin structure formation is there any trouble shooting method I can use , during primer design? I tried to design primer for a genomic sequence but could not avoid dimer formation. Please make a video regarding this.
Thank you for your question! You can avoid primer dimer formation by ensuring that there are no complementary sequences in your forward and reverse primers, and you can avoid primer hairpin formation by ensuring that you do not have 3 or more bases within the primer that complement each other. You can check for this when designing your primers by using primer design software such as OligoCalc (biotools.nubic.northwestern.edu/OligoCalc.html) or Primer3 (primer3.ut.ee/), which can check for self-complementarity in your primer pairs. I hope this information helps.
There are several companies that synthesize primers. We recommend viewing Sigma Alrich's website on oligonucleotide synthesis. Using solid-phase chemical synthesis, each nucleotide is added on the 3' end. www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/dna-oligonucleotide-synthesis
Hello, thanks for your question! The primer sequence is based on the template sequence (displayed in blue in the video). If that didn't quite answer your question, we suggest checking out Sigma Alrich's page on oligonucleotide synthesis, that may be of some help. www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/dna-oligonucleotide-synthesis
Thanks for the question! ddPCR first separates your reaction into thousands of individual droplet where each droplet is an individual PCR reaction. You can read more about it in the "AAV titering using droplet digital PCR" section of this blog post: blog.addgene.org/droplet-digital-pcr-for-aav-quantitation , or check out this page for morewww.bio-rad.com/en-us/applications-technologies/digital-pcr-real-time-pcr-qpcr-choices-for-different-applications?ID=OENHBB15
Hello, and thanks for the question. We're not exactly sure what you're specifically referring to, but if you have any questions, you can contact help@addgene.org and our science support staff can assist you.
Thanks for the question! An AT base pair has two hydrogen bonds, while a GC base pair has 3 hydrogen bonds so a higher temperature (4 C) is required to break a GC base pair, relative to an AT base pair (2 C).
![[TH] RNT vs SPG - VCT Ascension Pacific - Day 1](http://i.ytimg.com/vi/RUTkf8Detj8/mqdefault.jpg)
Addgene does not regularly monitor comments posted here, so we may not see your question immediately. We’d be happy to answer any questions sent to help@addgene.org as soon as possible. Please include the name of the video along with any questions so our support team can help. Thanks!
I like the way you explained everything with total calm and understanding, keep going and thank you❤
Thank you so much for this video! It's very helpful and explains the primers very well. And it'll surely help for my biotechnology exam next week^^
What are the online tools to warn us of potential hairpins, self-dimers, and cross-dimers?
Thank you very much! It is well-explained!! I never figured out the basic PCR design until I looked at you! You're awesome!! :)
Thanks! This is a super neat introductory video for primer design :)
Wonderful explanation! I hope you keep update.
Thank you sm. They just explained this to us in class and I didn't understand anything until I watched your video.
Complete information in the most simplified manner
My advice for y’all guys always use softwares when designing your primer will save you more time and much efficient .
what software do u use?
Thank you, Miss Ma'am! I love this video of yours, so comprehensive!
the content was great and so you were so sweet love it
Very informative and well-explained!!
Thanks Jennifer , That was both interesting and informative .
helps a bunch!!! very concise and clear!
This has been really helpful, thank you so much!
Thank you so much for this video.. it's very easy to understand..
4:04 put smile on my face :)
Thanks for this video! i gotta design primers for my project and this has helped me so much :))
Oh your happiness at the end😁🤗
What if you don’t know the DNA sequence? How do you choose primer?
It would be great if you could make a video about CRISPR KI... There is very little information available about this technique.
Thank you very much. I appreciate this video.
Omg, this is gold. Thank you so much:)
This is so simple way !! Thank you♥️♥️♥️
thanks it was really clear and helpful !!
Thanks. You speculated me with a standard presentation. I have one question if you don't mind. You said to add 4 degree for each pyrimidine base and 2 degree for each purine base to calculate melting temperature of the primer. Can you explain why I should add 4 or 2 in those perticular cases?
She explained later in the video, that C-G base pairs have 3 hydrogen bonds, while A-T pairs have just 2, therefore G-C interactions are stronger than A-T. hence the higher temperature required to melt G-C compared to A-T
GOOD LECTURE 👍GOD BLESS YOU TEACHER 😊
Thank u so much.. its so informative. Love it
Wow! Such a helpful video
Thank you so much it really helped a lot. I have my genetic engineering exams in few days. 😇😇😇✊🏻✊🏻
Very clear and informative
Best explanation!
Simple and easy to learn...thanks
Very helpful video, thank you teacher
I did that lil dance at the end with you 💃✨
Thank you for this video! Very well-explained (:
thank to you now i understand the basic,
nice.pretty good info,thanks
Lovely Information! Thanks a lot!
Thank you Jennifer
Very informative and interesting
honestly so helpfullll
great explanation , thanks
Thank you! So caring
I have the protocols for normal PCR.how can I optimize them for using multiplex PCR
These publications should be helpful :)
pubmed.ncbi.nlm.nih.gov/9298224/
www.sciencedirect.com/science/article/pii/B9780123721853500079
PubMedPubMed
good contents
hope you keep update :)
can i ask a question ? if you can't isolate a virus how can you design a primer for pcr ? you simply invent it ?
Thank you for your question. As long as you know the genomic sequence of the virus you are interested in, you can design a primer for that virus following the same tips and guidelines described in this video. You can also check out Sigma Aldrich’s page on PCR assay design for more information: www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/pcr-qpcr-dpcr-assay-design
Please tell me what are the reasons for the annealing temperature to be lower than the melting temperature by 5 degrees?
Thanks for the question! An annealing temperature 5C below the Tm can be used as starting point for PCR, but this temperature is something that may need to be optimized for high yields. A temperature of 5C below the Tm should allow for annealing to the expected target site. Lowering the annealing temperature further though could increase annealing to off target sites that differ by single nucleotide mismatches.
Very clear thank u ❣️
how do we find the tools to identify dimers and cross dimers?
Here's a link to a good resource that should help you with that: www.idtdna.com/pages/tools/oligoanalyzer
in case of primer dimer / hairpin structure formation is there any trouble shooting method I can use , during primer design? I tried to design primer for a genomic sequence but could not avoid dimer formation. Please make a video regarding this.
Thank you for your question! You can avoid primer dimer formation by ensuring that there are no complementary sequences in your forward and reverse primers, and you can avoid primer hairpin formation by ensuring that you do not have 3 or more bases within the primer that complement each other. You can check for this when designing your primers by using primer design software such as OligoCalc (biotools.nubic.northwestern.edu/OligoCalc.html) or Primer3 (primer3.ut.ee/), which can check for self-complementarity in your primer pairs. I hope this information helps.
Thanks for your video
Thank you very much.
Thank you doctor
So pretty ❤️❤️
Hi. Thanks for the video. :)
if we already get our design in software. how do we get the real one?
There are several companies that synthesize primers. We recommend viewing Sigma Alrich's website on oligonucleotide synthesis. Using solid-phase chemical synthesis, each nucleotide is added on the 3' end. www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/dna-oligonucleotide-synthesis
Question when making primers do you make them in a random order
Hello, thanks for your question! The primer sequence is based on the template sequence (displayed in blue in the video). If that didn't quite answer your question, we suggest checking out Sigma Alrich's page on oligonucleotide synthesis, that may be of some help. www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/dna-oligonucleotide-synthesis
Good video
helpful! and I like the background song/beats
Nicely eXplained
Could please explain ddPCR vs PCR. Thanks
Thanks for the question! ddPCR first separates your reaction into thousands of individual droplet where each droplet is an individual PCR reaction. You can read more about it in the "AAV titering using droplet digital PCR" section of this blog post: blog.addgene.org/droplet-digital-pcr-for-aav-quantitation , or check out this page for morewww.bio-rad.com/en-us/applications-technologies/digital-pcr-real-time-pcr-qpcr-choices-for-different-applications?ID=OENHBB15
@@addgene thanks a lot
How to upload the synthetic gene on the plasmids
Hello, and thanks for the question. We're not exactly sure what you're specifically referring to, but if you have any questions, you can contact help@addgene.org and our science support staff can assist you.
Well done!
Amazing!!!!!!
Thanks so much
Very nice 👍
I am definitely no longer amongst my reloading brothers. I’m lost
Very beautiful
I love the shirt and great video! :D
But primer are RNA segment not DNA.
They're DNA oligonucleotides, not RNA
Awesome!
why 4° and 2°C for the Tm ? :(
Thanks for the question! An AT base pair has two hydrogen bonds, while a GC base pair has 3 hydrogen bonds so a higher temperature (4 C) is required to break a GC base pair, relative to an AT base pair (2 C).
@@addgene thank you, you saved me!
I don’t know why I saw this but Jennifer is cute lol. And I learned something.
Very helpful vdo
can u answer the doubts in chat?
Thanks ❤️❤️
Todays exam studied🙏🏽😊
tnx a lot
Great👍
Super ❣️
pretty lady, pretty explanations.
👍🏻👍🏻👍🏻
Nice genes...
प्रेम
It bothers me how she keeps reading from the teleprompter.
No one cares about your opinion bro
@@felipevilicich980 reported for hate speech
@@cnsisow he called you bro. sounds more like a love speech to me !
@@cnsisow lmao how is them not caring about u hate speech
Thank you for the video
👍🏻👍🏻👍🏻