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Please direct all comments, questions, and suggestions to biobzc@hofstra.edu
SUBCLONING INTO AN EXPRESSION VECTOR
The expression vector that we use is pTrcHis2 (Invitrogen). This vector provides high level regulated transcription from the trc promoter and the lacO operator and lacIq repressor gene for transcriptional regulation of any E. coli strain. It also has a C terminal polyhistidine tag for purification and detection. Three vectors are available; each has the C terminal tag coding sequence in a different reading frame relative to the multiple cloning site to simplify in-framing cloning. Which vector should we use if we are going to digest the vector and the cDNA clone with EcoRI before subcloning? Many other commercially-available vectors would work as well. In this experiment we will be using heat shock transformation The pTrcHis2 cloning system uses blue/white selection to distinguish clones that are transformed with vector only from clones that contain the recombinant. Bacteria that incorporated plasmid with vector plus inserted DNA appear white on plates treated with X-gal (5-bromo-4-chloro-3-indolyl b-D-galactopyranoside) and IPTG (isopropyl b-D-thiogalactopyranoside). Bacteria that incorporated only the ligated vector will turn blue.
Since pTrcHis2 is a non-directional cloning vector, plasmids of the expected correct size (~9 kb, ~5 kb insert in the 4 kb vector) will be digested with XbaI and NarI to determine the orientation of the insert in the vector. There is a recognition sequence for XbaI in the multiple cloning site of the expression vector downstream of the EcoRI. insertion site. The Mhc sequence contains no XbaI recognition site. There is a recognition sequence for Nar I, 3.5 kb from the 5’ end of the Mhc fragment. There are no recognition sequences for Nar I in the vector.
Protocol for subcloning using pTrcHis2
1) Digest your cDNA and the vector with EcoRI. The Mhc transcript has two internal EcoRI sites, one at 979 which is before the start codon and the other 5bp from the stop codon. The cDNA can be digested completely since loss of either fragment is inconsequential to the expression of the gene.
2) Precipitate the digested clone and vector, spin down and ligate overnight.
3) Transform One Shot® cells by heat shock following the instructions provided by Invitrogen.
4) Incubate overnight at 37˚ C
5) Pick white colonies and grow overnight.
6) Isolate plasmid DNA.
7) Digest plasmid with XbaI and NarI overnight.
8) Run a diagnostic gel to confirm presence and orientation of the cloned cDNA. What size fragments would be obtained if the Mhc clone is oriented in the correct direction for expression?