Begin trimming bases away from the 3’ end of the sequence until the Tm is about 60 degrees. Remove the extra (non- complementary) leading sequence that you are going to add to the 5’ end of the gene as this will not bind to anything on the first PCR cycle: click analyze. Next analyze and trim the oligo: In the oligo menu click analyze. Save this sequence in the oligo list as’ oligo. Add the restriction site in front of your sequence with extra leading base pairs (e.g 3 extra base pairs for not1 to allow cutting don’t forget to add kozak consensus sequence in front of ATG for genes). Paste about 40 base pairs of the 5’ leading end of your sequence into the oligo list. How to design primers using Vector NTI: Enter sequence of your gene. Also ClaI in certain context gets dam methylated (see other protocol titled ClaI dam methylation). Also beware CMV promoter has an internal Nde1 site so this site will not be unique when working with CMV containing constructs. NB: Nde1 enzyme requires 7 base pairs in after the restriction sequence for efficient cutting. NB: Not1= GCGGCCGC and Kozak consensus sequence= GCC A/ TGCCATGG so a 5’ oligo for PCR of a gene to clone into position one should add NNN-GCGGCCGCC in front of the ATG start codon for that gene thus adding the Kozak consensus sequence with embedded Not1 site onto the 5’ end of the gene! (NNN= three random nucleotides in front of the restriction site to allow Not1 enzyme to cut efficiently. Positions for cloning: 1 st position: promoter: spe1/Not1ġst gene coding sequence (CDS): Not1/BamH1 Primer design for cloning genes into the lenti backbone: pdf version of this protocol as well as many other useful lenti and stem cell protocols.
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Also please refer to our CReM protocols page for a free downloadable. To design primers to add these sites to your cDNA, see the suggestions below. The plasmid backbone is modular with NotI and BamH1 sites flanking the first gene expression cassette (allowing any cDNA to be cloned into any pHAGE vector after attaching 5′ NotI and 3’BamH1 sites to your cDNA of interest). We have single promoter versions as well as double promoter and IRES-containing versions designed to accomplish single cDNA or dual cDNA vector expression. How to Clone into the pHAGE vector backboneĬloning into the pHAGE vector backbone is relatively simple. A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and cPPT is included in each vector to increase gene expression levels in transduced cells. This ensures that, after integration in the target cell genome, only the internal promoter of interest is active and no further viral replication or infection occurs. The engineered virus is replication incompetent and has a self-inactivating viral promoter (deleted U3). To summarize, the third generation lentiviral vectors employed in our studies are typically pseudotyped with a VSV-G envelope that allows stability and tropism for cells of multiple species including mice. We have previously published a detailed description of the lentiviral backbone and technique for packaging high titer virus for in vivo studies, and several of these protocols may be downloaded here by clicking on protocols. Kotton worked with the original pHAGE backbone during his postdoctoral fellowship in the Mulligan laboratory and the backbone has since been adapted to express the transgenes indicated on this page. The lentiviral system we employ is based on an HIV-1-based backbone, named ‘pHAGE’ (standing for plasmid HIV-1 Alex Gustavo George Enhanced) originally developed in the laboratory of Dr. PHAGE-EF1aL-TagBFP-W W=WPRE sequence L=long version of promoter Background on pHAGE: A third generation lentiviral vector These and other vectors are also available to Boston University Investigators at our CReM vector core webpage, please visit here for a more complete inventory.įor information on our iPS cell bank, please visit our CReM iPS Cell Core website. Kotton, serves on the Addgene Board of Directors. Some of our vectors are also available from Addgene, where our PI, Dr. pdf files by clicking on the relevant link below for each vector.
#Vector nti express for free#
Vector maps and sequences are available for free download as genbank formatted. Please visit our PROTOCOLS page for all protocols, including ESC/iPSC lung differentiation, lentiviral packaging and titering, or intratracheal lentiviral transduction of lung macrophages! Or take our annual hands-on 1 week course! Sign up through the CReM iPSC Core webpage. Requesting our cells is easy, click here for details!ĭifferentiating iPSCs in your own lab is a little harder, but we’ll show you how: Vectors, Protocols, and Cells So many cells, so little time….