I am trying to clone a plant gene in PYES2 NTC vector, After Transformation i got colonies I isolated the plasmid from them and did double digestion to confirm, I got correct size insert release in Two colonies. But when i did sequencing with vector specific forward primer, I got negative sequence twice. Why it so?
Hello, I suggest that you could test more clone to find the target clone or redo it from the beginning. To my understanding, you have double digest the gene segment and vector respectively with double endonucleases, and joined the gene and vector, then you did the cloning. So, the problem you faced now is illogical if your did the experiment correctly.
Can just first try sequencing of pcr elute to confirm expected transgene
yea, i think so. I usually confirm the size of insert segments by PCR and then sequencing them (about 5 clones) to find the right clone.
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