How to Make an SDS-PAGE gel

[svantiii]

Great work!

Thank you!

[mouseeatcats]

cool!!!!!

Check out our vid

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[VegasPartier]

i always have this problem where i introduce air bubbles when i add the stacking layer. i usually use a plastic pipette when i add the stacking layer. do you think this makes a difference?

is there a way to rid of air bubbles? would to the pt where it almost overflows be a good idea to get rid of air bubbles?

[labtricks]

@VegasPartier Yes, add the stacking layer until it almost overflows. Then when you slowly put in the comb, some of the mixture should overflow. And don't worry about the pipette tip - plastic is just fine! Good luck on your next gel =)

[tranceaddictallnight]

We use 8.8 pH TRIS for our stacking gel in our lab. What is the reason for the lower pH in yours?

[labtricks]

@tranceaddictallnight you want the pH to be about two pH units lower than your running buffer. In this case we are using Tris-glycine buffer for running the gel, which has a pH around 8.

What is the pH of your resolving gel? And what are you using as your running buffer?

[tranceaddictallnight]

@tranceaddictallnight we use 8.8 pH Tris for both resolving and running gels. what is the purpose for the lower pH in the resolving gel?

[alabonneheure1]

How come you gel does not leak? My gels leak all the time. I even use a round level to make sure the plates are leveled correctly. I Clean the plates with 70%ethanol and set them up but the gel still leaks. Is there a secret to aligning the plates? I even put some parafilm in the bottom part of the apparattus but the plates leak anyway. Any suggestions??

[labtricks]

@alabonneheure1 It's probably a seal problem. Have you seen our "How to Avoid a Leaky SDS-PAGE gel" video? (there is a link in the description box above)

Try the pen/microfuge tube trick. If you still have problems, let us know =)

[zileburki]

can you store the gel for later use?

[labtricks]

@zileburki Yes you can. Store the gel (inside the glass plates and with the comb) in a container, submerged in the Running Buffer that you use when running the gel. Cover the container with the lid. You can store it at room temp but I prefer at 4 degrees. Just remember to use the gel within a couple of days =)

[zileburki]

@labtricks Thanks :)

[zileburki]

@labtricks thank you :)

[zileburki]

@labtricks thank you :)

[zileburki]

@labtricks thank you :)

[zileburki]

@labtricks thank you :)

[nana22124]

when you cast the resolving gel do you wait for it to solidify before adding the stacking gel?

[labtricks]

@nana22124 Yes. As you see in the video at 3:10, add a layer of isopropanol to keep the gel straight, and wait until solidified (about 15 minutes). Then add the stacking gel.

[boringgrass]

haha ur comb broke, just like mine, biorad needs to make the comb stronger

[TheLiona84]

thanhs a lot:)  @labtricks

[TheLiona84]

how m time we should wait for running gel solidification, and then we add thestaking gel?thx

[labtricks]

@TheLiona84

It can take from 15 to 45 minutes, depending on what percent gel you are making, and how much ammonium persulphate and TEMED is added. An indication that your resolving gel is ready is when you see a clean straight line form at the gel-isopropanol interface when the gel is ready.

[labtricks]

@TheLiona84 and yes, after it solidifies, pour off the isopropanol (or whatever you are using) and then add the stacking gel.

[TheLiona84]

plz can we use pure water in place of isopropanol?and what is the diference?

[labtricks]

@TheLiona84 You can use water but be aware that water can slightly dilute the gels. So if you are working with a high molecular weight protein (the band appears near the top of the resolving gel), you don't want to use water because a gradient can form at the top of the resolving gel, and the protein will not separate properly.

So you can use water, but if you have a high molecular weight protein, I would not recommend it.

[ishanrathore]

thanks @labtricks

but even when we use single gel (native PAGE) or also agarose gel then also buffer is used instead of water

[labtricks]

@ishanrathore for conductivity. Distilled water acts as an insulator, therefore electrolytes are required to allow the current to flow. These are provided by buffer!

[mozhistmk]

Thanks a lot!!!

[ishanrathore]

why are buffers used in gel preparation & not water ? plz with detailed reason

[labtricks]

@ishanrathore well what is a buffer? Buffers provide us with an environment that tries to keep a consistent pH. So here we are using two different buffers: pH 6.8 for the stacking gel, along with pH 8.8 for the resolving gel. If we didn't use buffer, then the protein samples would migrate at the same rate in both the stacking and resolving gels.The wikipedia article on SDS-PAGE provides more detail on how this works (sorry can't post the direct link here, YouTube won't let me).

[ysjl03]

Awesome vid!! the plates and the casting frames looks exactly like the ones we have in UBC...

[ysjl03]

Awesome video!! the plates and the casting frames looks exactly like the ones we have in UBC...

[pkgem]

Great video. Very helpful. One question: Do you add neat isopropanol or a solution - if a solution what ratio of alcohol to water do you use? Many thanks.

[labtricks]

@pkgem we use neat (pure) isopropanol, straight out of the bottle

[pkgem]

@labtricks Thanks. My method suggests another alcohol but I don't have that. I do have plenty of isopropanol so I'll give that a go. Cheers.

[labtricks]

oops! sorry i accidentally deleted @admarshall617's question when responding:

the question was: "how long does it take for the stacking gel and the resolving gel to solidify "

our response is: "It can take from 15 to 45 minutes, depending on what percent gel you are making, and how much ammonium persulphate and TEMED is added. An indication that your resolving gel is ready is when you see a clean straight line form at the gel-isopropanol interface when the gel is ready."

[yomolandia]

thanks!

[bakopala]

Cool gut a molecular biology lab tomorrow and this was handy

[poxyratarsed]

AHHH! You make it look so easy! When I loaded my resolving gel, it turned out slanted and I couldn't use it :(

[labtricks]

@poxyratarsed Did you remember to add isopropanol? That is what makes the resolving gel straight =)

[MartinOsth]

@poxyratarsed

alternatively you could add 0.1 % SDS on top of the resolving gel when casting...

[34psychotic]

WOW thanks :) I'm doing my first gel tomorrow... it doesn't look so confusing now

[c00lwatter]

Yes, very helpful. This isn't very hard as compared to a lot of thins, but I am always worried that I will screw it up.

[prxsiempre]

it's a fantastic method... It was vey helpful...