Hi, please i need a help. Attached is my Agarose gel containing ligation reaction (pET28a) and VEGFA (insert).
After ligating for overnight at 16 degree and run gel with 10uL from the ligation reaction.
Here is what i got.
Lane 1: Uncut vector pET28a
Lane 2-3: VEGFA insert 1
Lane 4-5: VEGFA insert 2
What could that suppose to mean?
Thank you.
Did you not utilize a DNA marker as a reference for determining the size of DNA? Having a DNA marker would have simplified the process of confirming whether the observed DNA bands correspond to the unligated vector and insert. One approach to achieve this is by employing PCR amplification, using the ligated product as a template and employing primer pairs specific to the vector, gene, or a combination of both. In such a scenario, a smaller quantity of the ligated product would be required. It is advisable to consistently include DNA markers in agarose gel electrophoresis for precise analysis of results.
Thank you @ Usha. I did not include DNA marker. But i will do it in the experiment. I am trying to repeat the whole process. I will share with you how far it goes.
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