Matt Gethers/CRI, Thailand/Mayuree Lessons

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Mayuree Lessons

Primer Design

When adding bases downstream of a cloning site for a PCR Primer for better cutting efficiency, you can choose bases that are part of the template so that subsequent to the first amplification, there is greater specificity/higher annealing temp between primer and template.

Generating Knock-Outs

Single Recombination - Using a plasmid with the homologous region of interest, the entire plasmid integrates into the target genome, origin, antibiotic resistance, and all. This strategy can be problematic due to the polar effect (see below).

Polar Effect - When multiple genes are regulated by a single upstream promoter (i.e. polycistronic transcripts), a single recombination insertion of a plasmid containing antibiotic cassettes (or any other genes) with their own promoters can cause an upregulation of genes downstream from the insertion site if the plasmid promoters are in the same direction as the genomic promoters or a downregulation of downstream genes if the plasmid promoters are in the opposite direction as the genomic promoters.

Double Recombination - Only a fragment of the plasmid integrates into the target.

SacB - Produces a bacterial toxin - is useful as a means of negative selection.

Cre-Lox system/Frt-Flipase - Means of creating deletions in genes. Place an antibiotic cassette (or whatever you want, really) on a vector; have the AntiB flanked by two lox sites and have the lox-AntiB sandwich flanked by homologous sequence up and downstream of the gene target. This construct will integrate into the target genome. When it does, express Cre which will remove everything between the lox sites and religate. The result is a deletion rather than an insertion/disruption.

Blue-White Complementation/Selection

The LacZ gene encodes β Galactosidase which can be functionally and physically broken into two parts, the α and ω subunits. Only when coexpressed can β Galactosidase catabolize galactose (and x-gal) into lactose and glucose. The ω subunit is usually retained on the chromosome while the α subunit is encoded on a plasmid. To do a Blue-White selection, you can place a fragment within the plasmid-encoded α subunit. If the insert remains, transformed cells plated on x-gal will be white while plasmids that no longer have the insert will be blue.