Prior running on 200V for 2,5 hour on 4 degrees, 2 microliters of PCR products mixed with 8 microliters of denaturing buffer was treated at 92 degrees for 5 minutes followed by 5 minutes incubation on ice. Can anyone comment on the photo, what went wrong? Is it a denaturation or staining problem? Thank you.
Hello Hančević
What is your gel percentage? From your gel images I can see the staining was good but wells could be improved. looks like you need much more time for electrophoresis because your PCR bands are on top of your gel. You may need another 5 to 7 hours. I remember for my gels, in some cases I was waiting around 9 hours for electrophoresis! My gele percentage was 8% (bis to acrylamide ration of 1 to 29).
Good luck
Good points from Ali Javadmanesh and I agree with him.Also what size is your pcr product and you often get better results with added glycerol in the gel.What is your denaturing buffer....there is always a lot of DS dna in an sscp gel but that picture really does look like a lot of Ds dna.
Hi Hančević,
Based on my experience, your gel needs more time for running based on the length and conformation of your amplified sequence. In addition, adding glycerol only affect in higher temperatures (room temp. and above). As Paul Rutland said, you see only double stranded DNA in your gel. Give your next gel more time to run and you will see single stranded DNA also!
Thank you for comments and suggestions. We will try with longer electrophoresis.
until you get it working well you might try increasing the amount of SS dna by using asymmetric pcr
This may arise through the use of old running buffer,Try to change running buffer solutions each time you run the sample.Another thing use the correct amount of agaros during preparing the gel.Run into gel with appropriate loading buffer, Let, If you use 6x loadings buffer, you need to use 5 uL for every 1 uL sample volume.
Thank you.
Try to denature at 95 degrees for about 10 minutes, then keep in ice till you load the gel. Run at 50V or 100 volts till dye trails to bottom. 50V helps in clear band separation where as if you run at 100V the single bands will be together.
Hope this helps!
Hančević Katarina
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