Cloning from a pGEMT Easy vector - (Sep/16/2011 )
I am new to cloning.
my lab has a stock of a particular gene in pGEMT easy vector, but now i have to express that gene in certain cell lines.
As i know, pGemtT cannot allow me to express the gene in any cell lines (I hope i am right), does it mean that i have to choose two restriction enzyme sites in the pGEMT vector and cut it and ligate to another expression vector say pcDNA3.1.
I am just wondering if i do this, will the sequence between the insert's translational start site (i.e. ATG) and the Restriction enzyme cut site affect the expression of protein? In addition, should i add kozak sequence in front of the translartional start site in case i am using pcDNA3.1?
Yes, you need to clone the gene of interest from pGEMT into an expression vector like pcDNA3.1 in order for your GOI to be expressed in cell lines. Just make sure you cut out the insert that includes ATG and stop codon, and ideally there are some kozak sequence in front of ATG. You can also add kozak sequence using PCR cloning if there's none in pGEMT.
Always make sure you clone the GOI in the right orientation in the new vector.
THANK YOU very much for CloneMAN to reply!!
One more question.....if i want to add kozak sequence in front of ATG of the gene of interest, should I decrease the annealing temperatue during PCR to allow for the primer with the kozak sequence in front to bind on the gene of interst. Since the upstream of the gene of interst does not contain any kozak sequence in the plasmid, i am just worrying if the primer with the kozak sequence at the 5' end can bind or not and amplify the gene.
As long as you have enough primer sequence that anneals to the 5' end of the coding sequence (20-24 bases), you should have no problem adding a restriction enzyme site and a Kozak's seuence upstream of that. For example, the last gene that I cloned into pcDNA3.1, I used the HinDIII site at the 5' end, so my primer had a 5' tail:
5' - AAGCTTCCACCATG-<24 bases of coding sequence>-3'
I performed the PCR, cloned into pGEM-T, sequenced my clone to be sure it was correct, and then digested with HinDIII and another enzyme at the 3' end to cut out the piece from pGEM-T and ligate it into pcDNA3.1.
Best of Luck.