I have tagged pEGFP_N1 with APOE insert. However, after RE digestion, at gel electrophoresis, I am getting the insert band at wrong size. I have used EcoRI and SalI restriction enzymes. What could be the reasons behind this? I have tried twice and the result was similar.
I have used this plasmid. I wanted to know where the target gene was expressed within the cell. Ligation and follow-up would not be a problem if double digestion of the enzyme was not problematic. Alternatively, universal primers can be chosen near polyclonal sites on the plasmid for simple sequencing.
Dear Luwen Zhang
Thank you for your time.
Yes, I have used universal primers for sequencing. And to me, double digestion part was okay. Still searching the cause.
Hi there,
What do you mean by wrong size? How different are the expected and obtained sizes?
PCR diagnosis using plasmid specific primers is also a good screening method prior to sequencing.
Sorry, I did not quite get your question. There is nothing wrong with this genetic selection of the two restriction enzymes, and I have compared them. You have the problem that the plasmid after the gene insertion is re-digested and has the wrong fragment size, right? So the results after both end sequencing (CMV F, SV40 R) show that your insertion is not the length of APOE? Is this plasmid purchased as a finished product? Is there something wrong with the previous insertion?Or do you digest the empty plasmid and then compare it to the undigested plasmid, and there is a difference in size? I hope your experiment goes well.
Hi again,
Is the fragment 2kb bigger or smaller than expected? And what are the actual size of vector and insert when digested? A picture would really help us to help you...
There could be several reasons why you are getting an insert band at the wrong size after restriction enzyme digestion. Some possibilities are:
It's always a good idea to confirm the identity of the bands you observe by sequencing the insert to verify its size and identity.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
Thank you Nikolay Klyashtornyy for pointing out every possible way.
I will be indebted to.
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