You. Are. Awesome. You just explained something that two profs and 2 TA's failed to be able to explain to me in such an understandable way. Thank you!!
Thank you so much for this!! I looked everywhere on the internet and for some reason, the way you worded it specifically and drew everything out just made so much sense to me!! Thank you!!
Great video! My only comment would be to be a bit more aware of what can be seen on screen at a given time, as sometimes things were a bit cut off at the edges.
It's nice explanation. FYI: I understand it's for better explanation, but the template you choose is quite short isn't it, so that primers actually overlap. I doubt these primers will work because they form dimers too easily.
At 11:50 you say :"This low temperature annealing G and C means that when the temperature is increased in PCR this primer physically can not bind...." It is not correct. The primer with higher GC-content required higher melting temperature.
Thank you!!! I understood it perfectly. And guys, If you have SOME idea of basic genetics you wont need the view of the whole screen... Greetings from argentina!
It's simply when you are buying and ordering your primers from somewhere. Because the structure will be different if you go from 3' to 5' and 5' to 3' and if you ordered it as 3' to 5', you'll be getting a 5' to 3' primer from the vendor and it won't work as you imagined it to do.
excuse me will u plz tell me that if i have a DNA sequence of 3819 bp and i want to make primers from this sequence then can i start from anywhere in the sequence or just for fwd primer i would have to start from begining and for reverse i would start from the end of the sequence??
Are you saying that you have to reverse compliment the reverse primer or that you just have to reverse the sequence of the reverse primer? I'm asking because when I put in the original reverse primer that's in the 3'-5' orientation into a reverse compliment calculator I get the complimented sequence of your reverse primer and not the sequence you say is the reverse compliment.
If you turned the molecule upside down so that the top strand was the bottom strand, then you could read that strand left to right correctly. The problem is the way we lay the molecule on the page. The bases are complementary but because the molecule is laid out like that we have to read the base sequence backwards to make it forward. notice that the forward primer can be read forward simply because we are reading it left to right.
Where could I find explanation on what happens when primer binds to a different region of the plasmid with exact nucleotide sequence? How then that regions not get amplified to the comparable amount as the region of interest?
Hello Herbert, First off, thanks for doing this tutorial, great help to me. I wanted to ask, why not just make it 18 base pairs because there's already a G assigned on the 18th position. Is there a reason for not doing the suggested?
Found this on the internet: Primer Length: It is generally accepted that the optimal length of PCR primers is 18-22 bp. This length is long enough for adequate specificity and short enough for primers to bind easily to the template at the annealing temperature. Hope that helps!
If you want to splice two pieces of DNA to each other you can do it by stripping away a few bases from the 5' ends of both (assuming they are complementary ) and they will tend to ligate with a little help.
This video would have been significantly more helpful if we could have seen everything on the screen you were pointing to. On our end, your pointing and talking about something that is a couple inches from or visible screen. Thanks anyways though.
