Dear, all
Who can provide me with a detailed protocol for the realization of this electrophoresis? Or a protocol for the use of cyber green gel?
I have to carry out an electrophoresis on agarose gel to check if the amplification is positive, unfortunately I am not visualized, even the size marker does not appear under UV.
Can BET (Ethidium Bromide) be the cause? I used an old stock!
Thank you
in my opinion BET is not a problem but you can easily check the BET by adding it directly to the marker and run electrophoresis, and do not forget to add a dye like bromophenol blue or Xylene cyanol or both.
Gel pre-staining
Boil the agarose in buffer to dissolution using a microwave or heating appliance.
While still fluid, add 1 µL of the 10,000× SBG solution in DMSO per each 10 mL of gel solution. Mix thoroughly pour the gel and let it solidify.
For best results, add 1 µL of of the 10,000× SBG solution in DMSO per each 10 mL of buffer near the anode ("+", red wire). Run the samples.
Or
Sample pre-staining
Least sensitive, most economical method.
Mix 25 µL of DMSO and 1 µL of the 10,000× SBG solution in DMSO.
Add 1 µL of the solution to each sample to be separated on an agarose gel
Run the samples.
Can you see nothing by eye on your gel.Sometimes transilluminator systems will not show a proper gel picture if the filter is wrong, the settings ( too short an exposure time) or if the shutter on the camera remains closed. Another effect is photobleaching where if you look at a gel for much too long the image fades because too much UV light has been used, If you see size standard and bands but they disappear before you can take a picture then bleaching is probably the problem . Are you getting background fluorescence from your gel even though you are not seeing bands?
You may also try 1.0% agarose gel including 0.5µg/ml of GelRed nucleic acid stain.
Best of luck!
Agarose gels are commonly used in concentrations of 0.7% to 2% depending on the size of bands needed to be separated. Mix agarose powder with 1xTAE in a microwavable flask or heater. when agarose solution cool down to about 50°C added ethidium bromide to a final concentration of approximately 0.2-0.5 μg/mL. for example 13 microliter of ethidium bromide ( 1:10) in 30 ml agarose solution is needed to o visualize the DNA under ultraviolet (UV) light.
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