Restriction Cloning

[@QuinnTaran]

High quality lecture, accessible to non-experts like me! As someone who avoided molecular biology but now find myself steeped in it in my doctoral program, this is such a great starting point. Thank you!

[@gregorysmith1134]

Thanks to your lectures I am picking up molecular biology really well, better than I ever thought I could at my age. Please don't stop making videos. You are helping educate the world in a complex field of study.

[@GarciaVailahi]

Awesome lecture, thanks for simplifying a complicated subject

[@SamsonAruna-pp2se]

You are a life saver. You explanation made it look like a simple thing to know.

[@ikennaozor2313]

Thank you so much for this. My advisor for my Master's program told me to make primers for GFP tagging and thanks to your video I think I'm ready to design some!!

[@eliotthugh8894]

Fantastic video! I am just recently entering the field of molecular biology and your videos help me a lot in understanding the basics and basic experiment planning! Thank you so much

[@deemaalobaidi2110]

shouldn't the primer be complementary to the gene of interest?@11:43

[@katharinehubbard5043]

No - the primer is the starting point for elongation, ie the primer is physically incorporated into the new DNA strand. That means if you want a new copy of the gene of interest your primer must have the same 5’ -> 3’ sequence, not the complementary sequence. Hope that helps

[@mrighakalra6273]

Finally found a great video to help me clear out so many things I was confused with... Thanks a lot mam 😇😇

[@professorarshadmajid8475]

Excellent video. Very, very well explained.

[@LeonardoBoroni]

you saved me with this amazing video: clear and complete!

[@patrickrehorst6530]

Comprehensive explanation!

[@AnnalisaDAvola-p3j]

Thank you for your lectures. they are amazing. I cannot find any lectures as good as yours. Can you make a video on ligase independent clonig?

[@sadiachowdhury2524]

thank you so much!! brilliant explanation with drawings. you saved my time !!

[@okpolihenry983]

Excuse me MA, I need tutorials from you regarding cloning engineering, o wow!!!!! am amazed with how u tot this so well, thank you so much . Can u do a video using Gibson cloning method .

[@mdzakariamorshed6778]

I really love the way u explained ….

[@phil1pd]

This was great, thank you so much!

[@taylormarkel1094]

THANK YOU SO SO MUCH YOU ARE AMAZING!!!!!

[@XinyunQiu]

Here is a general structure of a primer combining these components: 5' - Leading Sequence - Restriction Site - Gene-Specific Sequence - 3' When designing primers for cloning, amplification, or mutagenesis, it's important to include specific sequences that ensure the primer's proper function. Below are the key components to consider when designing primers for these purposes: The primer above includes: Leading sequence: GCG (3 bases to enhance restriction enzyme efficiency) Restriction site: GAATTC (EcoRI) Gene-specific sequence: ATGACTGACTGACTGAC

[@prakritirds]

So it is always better to do directional cloning?

[@Bornst3ll3r]

You are the goat ! (Greatest of all time)

[@positivevibes2087]

How do we write the leading sequence? if some asked to design a primer pair?

[@axospyeyes281]

could you recommend a company that can add your primer and stuff?

[@ChecedRodgers]

Thanks so much for this very helpful video. I have a question. If polymerization only happens in the 5' -> 3' direction, how are the "floppy ends" (leader seq. + restriction site) polymerized? I assumed that the polymerase would attach exactly at the point where the primer + template had formed a short 2-backbone sequence, and attach dNTPs from there. Maybe I misunderstood this.

[@katharinehubbard5043]

This all happens after DNA synthesis! The strands are synthesised as a double stranded molecule as usual. The restriction enzymes cut the double stranded molecule to give the sticky ends, and then a ligase sticks them back together. Only if the ligase reaction is successful can the plasmid be duplicated - any pieces with unmatched sticky ends won't be replicated. Hope that helps!

[@ChecedRodgers]

@@katharinehubbard5043 Apologies, I am not sure if I asked my question poorly or if I misunderstood your answer. These things are hard to describe without pictures. :-) For a toy example, suppose my GOI is ATG+AAA+TTT+TAG, and that I possess one double-strand copy of this. Let the strands be S and S'. Suppose I wish to attach a restriction site to only the left end while doing PCR. If I understand correctly, my forward primer will be leader sequence + restriction site + forward base pairs, so something like ATATAT + GGATCC + ATGAA. And my reverse primer is computed as the reverse complement of the GOI tail: TTTAG -> GATTT -> CTAAA. I imagine that during PCR cycling, first my forward primer will bind to S' at 5 base pairs only (leader sequence and restriction site are not bound, only ATGAA), and polymerase will complete it such that it becomes ATATAT + GGATCC + ATGAA + ATTTTAG. Let this copy be C. Complement strand C' does not exist yet. Then on the next PCR cycle, my reverse primer CTAAA binds to the tail of C, and polymerase completes the strand so that I now have C and C'. I was confused about how the leader sequence and restriction site in the forward primer would get a complementary strand, because in the first cycle of PCR the polymerase only copies from the forward base pairs on. But I forgot that C will get a complement strand during the second cycle of PCR. Have I got it right now, or have I still missed something?

[@RickSanchez-lf5jy]

Hi, LOVE this content, only I have a question, so should you then also use the same logic to mod the reverse primer? Very sorry if this is a stupid question. But please keep on making this kind of content! (one on SLIC and others would also be great, you make it very comprehensible)

[@katharinehubbard5043]

Yes you can (should!) mod the reverse primer as required - include a restriction site, but can also include other elements (eg if want to rate your protein) - I have a designing cloning primers video if you want more detail :)

[@kavitashirsath366]

When is random cloning used?

[@bernabethtendero7196]

I'm trying to clone a bidirectional plasmid. I would like to insert my insert in a reverse direction. Will there be a difference in primer design?

[@katharinehubbard5043]

Hi there - the basic principle will be the same, but you will need to (I) clone in two genes separately and (ii) be really careful about which orientation each gene is inserted either side of your bidirectional promoter. You need to make sure that both genes are inserted so they read from 5'->3' after the promoter, which means the two genes need to be on opposite strands of the DNA facing in the appropriate direction. Hope that helps!

[@mrighakalra6273]

Mam do we prefer Partial digestion with RE for the DNA used so as to avoid getting too many DNA fragments...? And mam , please would you upload a video about different types of Restriction Enzymes , would be really thankful to you mam

[@alifahimkhan]

wow.