11 replies to this topic

[Dr_Watson]

Hi everyone,

I had troubles when loading DNA into the wells of an agarose long gel. There is only enough buffer (TAE) on top of the gel to cover it, for quality purpose the gel isn't supposed to be soaked.
So I have DNA samples with loading dye, centrifuged to avoid any bubbles, no bubbles in the wells. The final volume of the sample is 18µL which is not too much for the wells (I'm pretty sure they could contain twice as much).
Sometimes when I load the sample I take out the pipette and 1 sec later the DNA goes out of the well as if it was "sucked up" and that makes the DNA in the next well spill out too!

I have no idea what's going on or what could be the cause of it. Could it be that there isn't enough buffer on top of the gel and the movement caused by the pipette induces the spilling?

thanks for any help, ideas, cunter-curses you could provide!

DrW

[Adrian K]

Did you make your own loading dye? Sometimes there is not enough or glycerol was not added at all into your loading dye which makes it "float" up...

I not sure how does the buffer have to do with the quality of the gel, and I don't understand why  the gel was not soaked.

[pito]

Dr_Watson, on 21 April 2011 - 07:45 AM, said:

Hi everyone,

I had troubles when loading DNA into the wells of an agarose long gel. There is only enough buffer (TAE) on top of the gel to cover it, for quality purpose the gel isn't supposed to be soaked.
So I have DNA samples with loading dye, centrifuged to avoid any bubbles, no bubbles in the wells. The final volume of the sample is 18µL which is not too much for the wells (I'm pretty sure they could contain twice as much).
Sometimes when I load the sample I take out the pipette and 1 sec later the DNA goes out of the well as if it was "sucked up" and that makes the DNA in the next well spill out too!

I have no idea what's going on or what could be the cause of it. Could it be that there isn't enough buffer on top of the gel and the movement caused by the pipette induces the spilling?

thanks for any help, ideas, cunter-curses you could provide!

DrW

Its hard to tell without seeing you do it.

A reason for example can be that you push down the pipette too much (further then the "first stop", like when you would want to empty the pipette completely).

Another problem could be that you just dont put the pipette deep enough.. another reason can be that you put it too deep and break the wells...
Or that you just dont use enough loading dye? BTW: you mention you centrifuge the samples with the loading dye, but when you use your pipette to place your sample in the gell, do pipette the sample up and down in the tube before placing it in the well? (to make sure the dye is spread nicely in the tube/trough the DNA ?


Have you tried it with a gell that is covered more (more buffer) and did you have the problem then? If not, then it might be that there isnt enough buffer and that the fluid just comes out because there isnt enough fluid covering your well (and what you put into it).

[hobglobin]

Sometimes it is attributed to remains of ethanol in the DNA sample. Perhaps you should try to dry the samples again or longer...

[Dr_Watson]

Quote

Did you make your own loading dye? Sometimes there is not enough or glycerol was not added at all into your loading dye which makes it "float" up...

I not sure how does the buffer have to do with the quality of the gel, and I don't understand why the gel was not soaked.

I didn't make the loading dye but know what's inside and there is glycerol. I have to add that I used that same loading dye several times before when doing regular small agarose gels, and never had a problem.
I have no idea about the buffer and the quality of the gel, but that's what my boss said and quite frankly he really doesn't like to be questioned so I just do it that way. I think he said it was the signal that's better or something... though I should try to soak it completely once just to check.

Quote

A reason for example can be that you push down the pipette too much (further then the "first stop", like when you would want to empty the pipette completely).

Another problem could be that you just dont put the pipette deep enough.. another reason can be that you put it too deep and break the wells...
Or that you just dont use enough loading dye? BTW: you mention you centrifuge the samples with the loading dye, but when you use your pipette to place your sample in the gell, do pipette the sample up and down in the tube before placing it in the well? (to make sure the dye is spread nicely in the tube/trough the DNA ?


Have you tried it with a gell that is covered more (more buffer) and did you have the problem then? If not, then it might be that there isnt enough buffer and that the fluid just comes out because there isnt enough fluid covering your well (and what you put into it).

hm good points and already thought about that (that is far from being the first gel I had to load )
so - I do not empty the pipette completely (afraid of any bubbles that could cause trouble), meaning I dont push further after the first stop.
   - I try not to put the pipette too deep or it could provoke the kind of liquid movement I want to avoid. So I put it in the well but just at the top so it doesn't touch the sample that is at the bottom of the well.
   - the loading dye being 6x, to 15µL of DNA I add 3µL of dye. I mix the dye to the DNA  before centrifugation, mixing after that would defeat the purpose (avoid bubbles in the sample caused when you pipette up and down).

We are supposed to only put 100mL of buffer on top of the gel, I add more than 200mL I think last time I didn't have any loading problems when I added 300mL of buffer. but again when loading several wells on the same gel with samples prepared the same way, and loaded the same way, some will spill out others won't, even though once it starts spilling out all the buddys around just follow up...

Quote

Sometimes it is attributed to remains of ethanol in the DNA sample. Perhaps you should try to dry the samples again or longer...

dry again? they aren't dry in the first place. Just basic miniprep preparation using a kit so column. Same as always, and as I said before never had a problem with the small gels.

[pito]

Dr_Watson, on 21 April 2011 - 08:38 AM, said:

   - the loading dye being 6x, to 15µL of DNA I add 3µL of dye. I mix the dye to the DNA  before centrifugation, mixing after that would defeat the purpose (avoid bubbles in the sample caused when you pipette up and down).

No it doesnt....

If you centrifuge it, your loading dye will be more at the bottom.
You can easly pipette the content of the tube up and down without it causing many bubbles.......
If you do not do this, its possible that some amount (the upperpart of the tube) will get lost when pipetting it into the wells.
A good handling of the pipette can make you mix it without causing any bubble!

I dont know how you pipette the fluid out of your tube, put if you do it by sticking the pipette into the tube at the bottom , you will first suck up the heavier (with much loading DNA) content of the tube and the not so heavy will be sucked in as last and thus wil be put as first in the well...

If a not so heavy fluid is in a well and you push furrther down on your pipette to empty out the more heavier stuff, you can indeed lose the less heavy dna.. because it gets "pushed" up by the more heavy substance.
+ you will also lose a part of the loading dye because this is at the top of your pipette and will not be pushed into the well.. causing loss of DNA to (since the DNA has to be heavy enough (with the dye) to stay in the well...)

I know this sounds crazy and it will only play a minor role, but it does play a role...
I am nitpicking here.. but it could play a role.


Mixing the content of your tube is essential to have a good mixed DNA sample..

BTW: you centrifuge the sample, thus you must have seen this before: darkish blue at the bottom of the tube and more clearer at the top?

Anyway: this is nitpicking and also possible not important for you sine I dont know how long you vortex your sample... But if you do vortex it in such a way you see what I described about the darker blue at thebottom and the clearer at the top.. you should mix your samples before putting them on the gel. If not: you might lose DNA + see what you described: leaving of the clearer blue dye out of the wells because its pushed away by the more heavier darker blue dye).

Edited by pito, 21 April 2011 - 11:03 AM.

[lab rat]

To be honest with you Dr. Watson, I have never centrifuged my DNA samples before loading on a gel.

In the past, I ran my PCR, dropped the dye on a sheet of parafilm, then mixed the sample on the parafilm (if few samples), or dropped the dye directly into the PCR plate, then shoved it down into the sample using the multichannel & pipetted up and down a few times. Now I use GoGreen, which has the dye in it, instead of plain 5X buffer. You just pull the plate out of the thermal cycler and load the gel.

If the sample is forming a "plume" as you withdraw your pipette, try holding the pipette a little higher and not IN the well. If you're too deep, you are pushing the pipette against the bottom of the well, building up pressure as you dispense sample that can't exit the blocked tip, and causing the sample to jet out as you withdraw. If the there is sufficient glycerol in the sample, you can hold the tip above the well and it will drop straight down. If it floats out after loading, Pito and Dr. H's advice applies. Sometimes it helps to hover a microsecond longer to make sure all the sample has left the tip before withdrawing. Otherwise you could be trailing the very last of the sample as you move.

[Dr_Watson]

Quote

I know this sounds crazy and it will only play a minor role, but it does play a role...
I am nitpicking here.. but it could play a role.

not crazy at all, at this point I'm open to any suggestions
So after centrifugation there aren't two phases, which as you said would mean that the dye is at the bottom and would need to be mixed. But I can't be  100% sure that it is well mixed. However, I had that problem before when mixing the dye to the DNA and without centrifugation.

Quote

To be honest with you Dr. Watson, I have never centrifuged my DNA samples before loading on a gel.

Neither have I until two weeks ago. And because it was a suggestion made to resolve the spilling problem. Obviously it didn't help much.

So to resume, I should be careful about the dye being well mixed to the DNA, the pipetting and maybe add more buffer on top of the gel.

I'm going to check it out next week and will let you know if it helped.

thank you all for your replies and if you think of anything else, just let me know

have a happy easter, or Bonnes paques or whatever good weekend!!

Edited by Dr_Watson, 22 April 2011 - 05:19 AM.

[grvsomani]

keep the comb at least 2-3mm up from your gel tray. so that it not spread to other wells due to bubble or breaking of well due to tip

[phage434]

Smell your DNA and see if there is ethanol in it.  That would be my first guess.

[lab rat]

Dr_Watson, on 22 April 2011 - 05:19 AM, said:

thank you all for your replies and if you think of anything else, just let me know

have a happy easter, or Bonnes paques or whatever good weekend!!

You too, and bonne chance!

[Dr_Watson]

So latest news, I've made a new gel concentrating on the buffer. Which means, I used the same amount of DNA, same loading dye, not even bothering to centrifuge the samples this time. So as I said I focused on the buffer, adding at least 300mL of buffer on top of the gel and this time everything was ok. DNA stayed in the wells.

so just letting you know