basic gel question - (Oct/29/2006 )
Hello all,
I am having a problem pouring a 0.08% agarose gel, though I am following all the techniques correctly. This is how I was trained, but I am having problems doing it myself.
I am making a minigel, which contains 30ml of agar solution. I just make it up to 35ml and this is how I was taught (to have a little extra ml). For a 0.08% agarose gel, I weigh out 0.03g of agar and put it in 35ml of 1X TAE buffer in an Erlenmeyer flask. This is heated up for 30 seconds & I watch to see bubbles form in the flask. It is pretty hot when I take it out of the microwave. I let it cool down to room temperature (or until Iwarm where I can pick it up). Then I pour it into the minigel + comb apparatus. I waited for about an hour yesterday to let it harden, but it was still a liquid form when I came back.
I have another gel someone else prepared, but I'd really like to prepare & run my own. Is there anything that I am doing wrong or can be improved on? I think it is basically the heating step, though for the life of me can't understand why it's taking so long for the agarose to harden (it is agarose for gel electrophoresis).
If anyone had this problem, please let me know!! Thanks.
I am having a problem pouring a 0.08% agarose gel, though I am following all the techniques correctly. This is how I was trained, but I am having problems doing it myself.
I am making a minigel, which contains 30ml of agar solution. I just make it up to 35ml and this is how I was taught (to have a little extra ml). For a 0.08% agarose gel, I weigh out 0.03g of agar and put it in 35ml of 1X TAE buffer in an Erlenmeyer flask. This is heated up for 30 seconds & I watch to see bubbles form in the flask. It is pretty hot when I take it out of the microwave. I let it cool down to room temperature (or until Iwarm where I can pick it up). Then I pour it into the minigel + comb apparatus. I waited for about an hour yesterday to let it harden, but it was still a liquid form when I came back.
I have another gel someone else prepared, but I'd really like to prepare & run my own. Is there anything that I am doing wrong or can be improved on? I think it is basically the heating step, though for the life of me can't understand why it's taking so long for the agarose to harden (it is agarose for gel electrophoresis).
If anyone had this problem, please let me know!! Thanks.
It seems to low percentage to me... Could it be you want to prepare a 0,8% gel instead of 0,08%?
Exactly. 0.08% "gel" is likely to be a liquid. The useful range is 0.5 to 2%, and rarely above or below those percentages.
I agree with DNAfactory and phage, 0,08% seems very low. I never saw such a low percentage before.
Maybe it's 0,8% ?
Hello,
Yes all. Sorry, that is a typo on my part. It is a 0.8% agarose gel.
Does anyone have any clue?
Yes all. Sorry, that is a typo on my part. It is a 0.8% agarose gel.
Does anyone have any clue?
Then you should weigh 0,3g
Hello,
Yes all. Sorry, that is a typo on my part. It is a 0.8% agarose gel.
Does anyone have any clue?
Then you should weigh 0,3g
Hello,
Yes, it is 0.3g. I was weighing out 0.03g. What a goof! But the gel worked fine with 0.3g + 35ml 1x TAE buffer and my DNA showed up beautifully! A happy ending.
Thanks all.
Hey that was a nice beginning and a good ending