Two days ago, I carried out the Agarose gel electrophoresis. And also, I performed the PCR prior to the electrophoresis (bt_3294 [KCTC 5015]). (The process that I did is on the picture.)
But I got some problem with the electrophoesis. I got a normal band at 'a' and 'b', however I cannot get the band at 'c'.
At first, I thought the primer did not function effectively during the PCR. However, if that was the case, there would likely be a primer dimer band present at 150-200bp.
So here is the question.
1. Can the band not appear even if the primer dimer occurs? 2. If there were any other causes, what could have caused it?
I will conclude this writing by wishing you a good day today.
Best regards,
Joonseo_Cha
Did you use the same concentration of template DNA? The large amount o smearing in lane A and B indicates quite a high concentration of template compared to lane C.
Getting amplification in other samples means the primers will create a product (at least for those templates). Is the DNA from the same source (e.g. all from mouse tails of BL6 mice) or are the DNA samples from different sources (e.g. corn leaf vs strawberry roots)?
Also, the multiple bands in the other lanes mean you have a lot of non-specific amplification. What size product are you expecting?
I suggest talking with a lab member or your PI to know what you were expecting for this part of your protocol.
Good luck!
the absence of DNA band in the selected strain may be due to
1- there is no genes for that particular trait.
2- may be you don't use the proper primer reconstitution concentration.
3- yo missed to add the template DNA.
Use a positive control in your experiment
I recommend all the above answers in addition check the voltage used in the electriphoresis process if your gene size is very small it could run out of the gell if the voltage is not suitable
I don't think there is any problem with electrophoresis as the gel image looks fine. Did you forget to add primers in the lane c reaction mixture? But I can see a faint band in Lane c. What's the size of your gene of interest? You need to give more information regarding the composition of the PCR reaction mixture used in all lanes. Are the templates used in all lanes different or different concentrations of the same template used? Also, there is non-specific amplification, you need to optimize annealing temperature and concentration of the template for better result.
Gel electrophoresis can sometimes result in faint bands or absence of bands due to incorrect sample preparations,low protein conc.,insufficient electrophoresis condition,or problems with gel or buffer
Concentration of gel is so important based on your product lengths
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