Restriction Digest Analysis

In this video, we will digest a plasmid of known identity with restriction enzymes, run the products on an agarose gel, and analyze the results. We’ll focus on identifying the bands produced under different conditions and use the results to make conclusions about the plasmid.
Note: XbaI is effected by Dam methylation - see this blog post for more information blog.addgene.org/plasmids-101-...
For the full written Restriction Digest Analysis protocol, visit:
www.addgene.org/protocols/dia...
________________________________________________________________________________________________
0:00 Intro
1:00 Determining Expected Results - Control
3:21 Determining Expected Results - Digestions
4:31 Run the Gel
5:40 Comparing the Data

[@addgene]

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 [e-mail] as soon as possible. Please include the name of the video along with any questions so our support team can help. Thanks!

[@romimoirangthem9277]

By far the best result analysis , explained it so easily

[@catherineespinoza5917]

This is very useful. I am sharing this with my undergraduate students to help them understand their lab results.

[@addgene]

That's great to hear Catherine! Please let us know if there are any other videos that you'd think would be particularly helpful.

[@milokenneth6303]

You prolly dont care at all but does someone know a way to get back into an instagram account?? I was dumb forgot my password. I would appreciate any tips you can give me

[@novelas3536]

@@milokenneth6303 Bro what

[@start5256]

You people are doin wonderful job great really crystal clear

[@nbent4607]

Direct to the point... I like it!!

[@hannahchen9272]

Thanks for uploading this video, it helps a lot in interpreting the results I got from my first 200-level biochem lab, and would be more confident about what we are going to analyse in the following lab.

[@addgene]

Glad to hear you've found it useful Hannah!

[@kat-thee111]

This is such a clear video and so well explained. Very easy to follow, thank you

[@GoldbergDaBoss]

This was great! You just saved my genetics lab report

[@addgene]

glad to hear! we also have more detailed protocols over at addgene.org/protocols/

[@crys1537]

Really helped me out for my internship thanks alot !

[@addgene]

Glad to hear you found it useful!

[@BoyMonkeyKid]

laughing while learning should be illegal

[@EatingWithEssence]

Great explanation!

[@hebaemam]

very helpful video

[@rj6110]

lots of helpful stuff here but im most impressed with the ability of the presenter to draw a perfect circle freehand lol

[@mohamedelmasry6388]

you're awesome ♥️

[@Hoxgene]

this was amazing

[@raselbarua4578]

useful video, keep going

[@mahdis.the.curious]

Having both nicked and supercoiled bands in lane 2 indicates that some of our plasmids stayed intact, and some others got nicked when undergoing the same conditions?

[@anjaligupta6180]

For Age1 and xba1 double digestion (lane 5) How much nanogram of plasmid DNA was required to see the 300 bp band on gel. ?? If i have my estimate Dna conc around 25 ng/ ul post boil prep.

[@hebaemam]

you are so funny guys

[@smilykhajuria4411]

Hy ..what is the procedure for ordering the plasmids from your site

[@bluebyrne7864]

Thank youu

[@henkdevries2002]

What is the song in the intro?

[@divyagautam4115]

If I have two bands showing in a single well during agarose gel electrophoresis, after RE digestion what does that mean

[@addgene]

Thanks for the question. If you observe two bands on a gel after a restriction digest with a circular DNA molecule, such as a plasmid, that means your restriction enzyme(s) have cut two sites on your DNA molecule, producing two linear fragments. If you see two bands after a digest on a linear DNA molecule, that means your restriction enzyme(s) have cut only one site on your DNA molecule, producing two smaller linear fragments.

[@CestlaBios]

How can I know the concentration of restriction enzyme needed for this characterization?

[@addgene]

The concentration of the restriction enzyme will be on the tube of enzyme, usually in "U/mL" or "units/mL." Usually using ~0.2- 0.5 uL of enzyme will work. For more details see this protocol: www.addgene.org/protocols/restriction-digest/

[@srinathkshultz9968]

why only thosetwo enzymes ? why not gowith other restriction enzymes ki ecor1 or hind III

[@addgene]

The enzymes shown in our video are for illustrative purposes, but within a plasmid sequence there are numerous restriction sites that you could consider using. On our plasmid webpages, unique restriction sites are annotated in bold on the plasmid maps. If you are performing a digest that is expected to return multiple bands, we recommend first confirming that these will be of sizes that can be easily separated on an agarose gel.

[@mukhodenimatodzi3844]

Univen students 👩‍🔬 where are u?

[@someguy1576]

Why those enzymes in particular?

[@addgene]

Hi, these particular enzymes were just used as an example. It'll differ depending on the sequence of your plasmid which restriction enzymes you should use to cut the plasmid.

[@srinathkshultz9968]

@@addgene what is the basis to choose those enzymes?

[@guihuiqin6679]

Cute~

[@ericfeldsott7481]

he's wearing shorts in the lab-- rookie mistake

[@zangetsu6638]

4:55 #metoo that dude is gone!

[@cinnamonbun216]

Not funny

[@zangetsu6638]

@@cinnamonbun216 and 5:11 too i think the dims have found biden's running mate

[@raunakgupta2148]

Oh well that was 2018 :, )