Problem Viewing bands in Agarose Gel
Posted 12 April 2011 - 04:02 AM
Moved to a new lab these days, I seem to have trouble viewing my PCR products in agarose gel electrophoresis.Having performed successfully this technique many times in the past, I really wonder what is going wrong without being able to answer.
The facts are the following:
1. Agarose gel 0.7%, made with 1X TBE
2. "Home made" TBE 5X (Tris Base 54gr, Boric Acid 27.5gr, EDTA 2.92gr in 1L ddH2O)
3. DNA ladder (100bp, BioWhittaker)accompanied by its own 6X loading buffer.
4. As tracking dye for the PCR products, I used either the loading buffer mentioned above or (for fear of it interfering wrongly with the PCR product) I used the Blue/Orange Loading Dye 6X (PROMEGA)
5. 7ul of EtBr (made by dissolving a 10mg tablet in 1ml ddH2O) in 100ml of agarose-TBE solution after melting thouroughly the agarose in microwave oven.
6. Running time and Voltage: until the dye reaches almost half the gel length and at 100Volt
Any ideas why both marker and PCR products seem not to exist???
Posted 12 April 2011 - 04:34 AM
Welcome to the Forum!
You have mentioned that you have worked with this earlier, but I would like to ask,
1. Do you add EtBr, right after the agarose is out of the microwave, or do you wait for a while, let it cool down and then add EtBr?
2. Have you considered adding EtBr to the buffer to see if you see any bands?
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Posted 12 April 2011 - 03:24 PM
Posted 15 April 2011 - 03:11 AM
Well, as for the EtBr, I do not add it straight after I get the beaker out of the microwave but I do add it certainly before letting the agarose cool down enough to be able to pour it in the casting tray. However, I now recall that I have stopped using something to cover the beaker while waiting for the cooling. ( maybe I have losses from there....)
Regarding the DNA, as it's from a new Enterovirus protocol, the PCR product is quite large and I use 10ul of PCR product plus 2ul of tracking dye (6x).
I'm thinking that I also have to face quite a few barriers such as the "antiquity" of the electrophoresis machine (it's really a bit old and while trying to keep the voltage set at 100Volt, 5 minutes later I find it dropped down!!!!!).....On the other hand I'm afraid this might be something that doesn't really matter...Does it?
Posted 15 April 2011 - 03:13 AM
Posted 15 April 2011 - 04:14 PM
Posted 15 April 2011 - 05:15 PM
What happens when you leave the EB out of the gel and put it only in the buffer? Does background from the EB still show up?
Edited by lab rat, 15 April 2011 - 05:17 PM.