I have digested the genome by EcoRV restriction enzyme, and then I amplified it for the target sequence in PCR. I am expecting a smaller band size from the amplification than the undigested genome template, but it resulted a nearly 10 times bigger band size. Therefore, I am thinking if the PCR amplification could re-ligate the cutting site again?
Why are you expecting a smaller amplimer after digestion Kayna Yong ? If the target sequence is between Ecorv sites I would expect the same size amplimer but if there is a cut site within the target sequence I would expect no amplimer. Am I missing some step in the protocol? My first thought is that the pcr could not work for your amplimer and that the large band is a non specific product
For ligation to happen,there should be an enzyme called ligase. This enzyme is the one to join and seal the two ends of your fragments. For your case, may be the restriction enzyme EcoRV was not effective in digesting your DNA like if the incubation period was reduced or concentration of the enzyme. This is what i can explan to my understanding
Thank you for your comment Paul Rutland. I never done this experiment before, so I think I must have mistakenly thought that the restriction enzyme will shorten the target sequence by cutting it. My cutting site is within the target sequence, so I should expect no amplifier. Is this because the site has permanently cut, and it interrupted the amplification process?
Thank you for your comment @Rule Budodo. I have incubated the genome with the restriction enzyme for 24h overnight and the concentration is 20unit to 1ug DNA (NEB protocol).
yes Kayna Yong if there is a cut between the primers then the Taq falls off and there can be no product. If you need a shorter product then you will have to redesign the primers closer together and amplify the undigested genomic dna
Dear Kayna Yong,
I think your digestion did not work properly. You may use EcoRV-HF restriction enzyme from NEB with rCutSmart Buffer. EcoRV-HF enzyme would be more efficient for the digestion as we as it will need less incubation time than the normal EcoRV.
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