Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

GFP vector - (Jan/13/2014 )



I need to clone a PCR generated insert into a GFP-tagged vector. Don't want too strong an expression of the insert. Goal is to transfect, select and use pooled population for an 8 day growth assay. 


My questions:

Can you help me with choosing a GFP tagged vector--which promoter?, should GFP be on the amino or carboxy?, guess for my purposes I don't need to use a lentiviral vector--right? and with respect to cloning--I would like to use a vector that allows for easy ligation of the sequenced insert--ie without restriction digest.


Thanks in advance!




The promoter you should use will depend on the end application and which organism (or which cells) you are wanting to use the vector in.  In mammalian cells viral promoters such as CMV and SV40 produce strong expression.  Human or mouse promoters produce much weaker expression. 


You should produce vectors with GFP at both termini, so that if the GFP interferes with the protein in some manner, you may be able to get around this with the GFP being on the other terminus.


TA cloning is the one you need to go for if you don't want to restriction digest. 


Finding all 3 of your requirements is unlikely - especially the TA bit.