Basic SDS PAGE problem..

[Gavanshir]

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Hello,

I'm having a problem loading my samples into the wells. I load my Kal marker and it's fine, then when adding my sample into any of the wells, they simply slowly diffuse out of the well and into the migration buffer! Can someone please point to my problem Thank you.

 

[146233]

Pre-Allo is hardly the forum for this question.

 

[Fiddlergirl]

You may not be inserting the tip of the pipette deep enough into the wells. Having said that, SDN is probably not the place to ask this question. If there's a TA or lab tech around they can see the problem much better than me.

 

[bola]

right off the top of my head...maybe there's not enough glycerol in your loading buffer, since that's what essentially weighs the samples down.

 

[Gavanshir]

right off the top of my head...maybe there's not enough glycerol in your loading buffer, since that's what essentially weighs the samples down.

I think this is probably the problem. I'm using 5X loading buffer and 5 ul of it. I think I should increase its volume.. thanks.

 

[nick_carraway]

Well you should be using 1:5 dilution of your loading buffer to your sample. If you're using 5 uL that means you're using 20 ul of protein?

 

[146233]

Well you should be using 1:5 dilution of your loading buffer to your sample. If you're using 5 uL that means you're using 20 ul of protein?

You're encouraging him!

 

[nick_carraway]

You're encouraging him!

I know... and now I feel terrible for bumping this back to the top. The mods are going to hurt me, aren't they...? 🙁

 

[146233]

I know... and now I feel terrible for bumping this back to the top. The mods are going to hurt me, aren't they...? 🙁

No, they're going to make you MEG@COOL's keeper.

 

[Gavanshir]

Well you should be using 1:5 dilution of your loading buffer to your sample. If you're using 5 uL that means you're using 20 ul of protein?

You are right, I wasn't using a 1:5 dilution. My total volume is 50ul and I was using 5 ul of loading buffer, it should be 10 ul. The volume of protein is calculated accordingly for each sample to contain 20 ug of protein. And the rest is H2O. Thanks.

 

[Noeljan]

Also
make sure you are watching your technique. Go as far down in the well as possiblle (without poking the gel of course). Also, release the pipette very slowly, and make sure to empty out the contents before pulling the pipette out of the well.