Our aim is to transfer gene from bacterial plasmid(pET22b) into mammalian expression vector pACGFP1-N1, i have decided restriction sites available in pACGFP1-N1 vector MCS but while doing restriction digestion followed by ligation i always got colonies in vector transformants and in vector plus gene transformation we do not got any insertion.
What does the no-ligase vs + ligase control look like? If you have same number of colonies without ligation, then you have background of uncut vector. If you only get colonies upon ligation then either you have some vector only singly digested and not double digested or else your insert is not so good so the frequency of ligation products is low. Did you screen a large number of colonies before saying you had no inserts?
Did you screen all of your colonies? Sometimes we need to screen a lot to find the desired one.
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