Hi! I made DIG labeled RNA probe for ISH through in vitro transcription. I am wondering why I saw 3 band on agarose gel. It is because polymerase amplified other form of DNA (supercoil,circular etc) which may be exist after linearization of DNA ? However, I tested my linear DNA there is only 1 band. I used that linear DNA to make a DIG labeled RNA probe. So how can I understand that 3 bands belong to my gene or non specific bands?
Any comment is appreciated!
Murat
thank you for suggestion. I am sure that my DNA is linear. (I tested linearization of DNA on electrophoresis with plasmid DNA as a control.
Did you heated up your probe (60ºC for 10 min) before you run it? If you didn't it's normal that you have multiple bands, that correspond very likely to secondary structure or dimerization of your probe.
Hope it helps!
thank you for your response! yes I heated probe before run gel. However, still multiple bands appear on the gel
1. Linearize DNA and gel-purify this template to remove all other species of DNA.
2. After IVT, digest DNA with RNAse-free DNAse. The only thing left will be RNA.
3. As previously noted, multiple bands can occur due to ssRNA dimers or secondary structure. You can avoid this by running a denaturing gel, but this step is probably not necessary.
4. If you get repeatable, distinct band(s) and not a degraded smear, your probe is likely good, and you should try it.
Good luck with your experiment.
Hi Murat
If you are purifying your linearized DNA by ammonium acetate: ethanol precipitation, then please check that you completely air dry the DNA pellet after washing. Sometimes residual trace of ethanol effects your IVT reaction.
Good Luck.
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