problem with agarose gel? - (Jul/27/2005 )
First of all I want to apologise for my little broken english because I am not a native English speaker.
I have a big problems with bands on agarose gel.
I send to you two pictures to see what my problem realy looks like.
For these pictures were speceification:
40ml TAE buffer(1x) , agarose 4%(1,6g), CybrGreen 0,5x, marker V(0,4 micro l). I maintain temperature of buffer 20 C(77 F) and voltage was 75 V for 2 and half hours!
Oh, yes. I try to separate bands(152bp and 170bp)-barely seen on picture.
Any idea, how to improve this picture, because now its unusefull. I try almost everything and work on this f... "project" the whole July. Sorry for the word with F. I am really angry with this.
Yes, I will ask you sth more about boiling the gel. Is it important to maintain rotation within boiling?
I couldn't find any troubleshooting with gel agaroze on net. Has anybody found sth?
Realy, realy thx for any ideas in advance!
Greetings from sunny Slovenia
Probably you have already tried/considered, but there are special types of agarose to use when making high percentage gels NuSieve GTG is the one we have. Maybe that will help...
Also, I have never continuously rotated the TAE/agarose when melting, I just use a regular microwave and take the bottle out every 30sec or so to mix (also to prevent boiling over)
One other thing that may help is staining the gel afterward. I have not used CybrGreen, but EtBr runs backward out of the gel and would be removed from the bands at the bottom of the gel simply because it is positively charged and runs to the opposite pole from the DNA In some cases my small DNA bands have "disappeared" and I can see again upon re-staining with EtBr
hope this helps, good luck
Hi there,
So you are trying to separate two band with 18bp difference, right? The problem is probably due to the 4% gel that your making. For a 20bp separtation a 3% works great (100V for 1 hour). Make sure the gel is well dissolved (clear liquid) and make sure you weight the container before and after heating the agarose and make up the difference with dwater.
How are you staing the gel, adding the cyberG to the gel during the run or staing after the run. Running with cyber in the gel can alter it's migration and I believe it's recommeded to stain the gel after a run. In any case there is too much added. Reduce the amount by half.
HTH
Jeff
Also, I have never continuously rotated the TAE/agarose when melting, I just use a regular microwave and take the bottle out every 30sec or so to mix (also to prevent boiling over)
One other thing that may help is staining the gel afterward. I have not used CybrGreen, but EtBr runs backward out of the gel and would be removed from the bands at the bottom of the gel simply because it is positively charged and runs to the opposite pole from the DNA In some cases my small DNA bands have "disappeared" and I can see again upon re-staining with EtBr
hope this helps, good luck
[quote=jeng,Jul 27 2005, 12:58 PM]
Hi there,
make sure you weight the container before and after heating the agarose and make up the difference with dwater.
[/quote]
[/quote]
Jeff,
Why do you use water to make up the volume lost during boiling? why not TAE??
We use a lid when boiling anyway, this should help with evap. so you wouldn't have to do the above right?
One other comment, I got a good trick from someone... You boil the gel in 1/2 the volume TAE then bring up to full volume after agarose is melted. This saves any evaporation problems, and cools your gel to the perfect pouring temperature immediately after melting. It has worked well for me.
How old is your TAE?
are they fresh preparations? also are you using fresh TAE in the tank?
I found that old running buffer is the cause for the funny "bands" I have every now and then. They look similar to the ones posted here.
I don't think it is the agarose per se that is giving you such poor bands. I suspect it's the running buffer, either it is not fresh or made up incorrectly.
Good luck!
Nick
Hi there!
Thx every one for discussion. Today I will try:
- not boil over my gel and use water to make up the volume. Until now I have just put the cap of glass when boiling my gel and thats it. So I will try with this trick
- Yes, the amount of CybrGreen is to high, so I have reduced already. I put the CybrGreen soon after the agarose is hot and melted. Colud that be wrong? Tomorrow I will try with boiling the gel in 1/2 volume of TAE(thx beccaf22).
We had used EtBr until 2 years ago, but now in our lab don't want to use any mutagenic dye. Thats the reason I use CybrGreen. I used to do with CybrSave but it disappeared after 5-10sec.!! under UV light(312nm).
- and immediatelly I will channge the buffer. What does it mean, that the buffer has to be fresh? I change it every 10th run. Is that OK? More often? Less? (I maintain the buffer temperature usually 20C(77F))
Once again thank you for your coments and tricks. I will try and report my results
P.S.:Just for imformation, I am a student and working on polymorphism
According to my experience, there are some problems of your agar gel:
1:the slot of the gel is too bad, maybe you pulled comb out too earlier.
2:if you wanna separate ~150bp band,you should change your marker,that will be better.
3: lower the voltage of electrophoresis.
best wishes!
Thx every one for discussion. Today I will try:
- not boil over my gel and use water to make up the volume. Until now I have just put the cap of glass when boiling my gel and thats it. So I will try with this trick
- Yes, the amount of CybrGreen is to high, so I have reduced already. I put the CybrGreen soon after the agarose is hot and melted. Colud that be wrong? Tomorrow I will try with boiling the gel in 1/2 volume of TAE(thx beccaf22).
We had used EtBr until 2 years ago, but now in our lab don't want to use any mutagenic dye. Thats the reason I use CybrGreen. I used to do with CybrSave but it disappeared after 5-10sec.!! under UV light(312nm).
- and immediatelly I will channge the buffer. What does it mean, that the buffer has to be fresh? I change it every 10th run. Is that OK? More often? Less? (I maintain the buffer temperature usually 20C(77F))
Once again thank you for your coments and tricks. I will try and report my results
P.S.:Just for imformation, I am a student and working on polymorphism
Try low melt agarose if you are going to use 4% concentration.
Hey ,
SYBR green is known to alter the movement of DNA in the gel and distort it. I use SYBr green too....Consider using 1:10000 X dilution of SYBr ..ie 1ul/10ml of the TAE buffer ....u should use TAE and not TBE..althought TBE is a better buffer TAE is good for seperating small differences..also i do think that the DNA concentration is really high in the gels.....load lower volume of DNA if it is highly concentrated...
Thanks
Solution
Hi beccaf22,
Only the water evaporate not the salts. So if you making up evaporation with TAE than ithe concentration of TAE would not be 1x anymore 
k4bf0389, how's the gel?
Jeff
[quote=beccaf22,Jul 27 2005, 05:48 PM]
[quote=jeng,Jul 27 2005, 12:58 PM]
Hi there,
make sure you weight the container before and after heating the agarose and make up the difference with dwater.
[/quote]
[/quote]
Jeff,
Why do you use water to make up the volume lost during boiling? why not TAE??
We use a lid when boiling anyway, this should help with evap. so you wouldn't have to do the above right?
One other comment, I got a good trick from someone... You boil the gel in 1/2 the volume TAE then bring up to full volume after agarose is melted. This saves any evaporation problems, and cools your gel to the perfect pouring temperature immediately after melting. It has worked well for me.
[/quote]