Techniques Making a Crude Cell Extract

Why bother understanding how a technique works? Why not just do it? Because understanding: n Minimizes performance errors n Optimizes troubleshooting success n Provides starting point for making improvements

Making crude cell extract n Extract contains genomic (nuclear) DNA n DNA is template for subsequent PCR reaction

Source of cells n Follicle cells at the base of a hair shaft

Strategy for making DNA accessible as template for PCR n Lyse cells to release chromatin (DNA and associated proteins) into solution n Degrade the protein to remove it from the DNA. n Leave extract in a suitable form for the next step (PCR)

Critical reaction parameters for making cell extract n Buffer contents n Inclusion of proteinase K n Temperature of reaction n Time of reaction n Ending the reaction

Buffer contents - compatible with protease and PCR polymerase n Tris-HCl - maintains pH at 8.3 u proteinase K works at pH 8.3 u Taq PCR polymerase works at pH 8.3 n Salts mM MgCl 2, 50 mM KCl u Chosen to be compatible with PCR conditions u Proteinase K also works

More reaction components n Gelatin - denatured collagen u Raises total protein concentration in reaction u Higher [protein]  stability of enzymes n Detergents u Lyse cells and dissolve nuclear envelope F Release chromatin (DNA + bound proteins) F Expose DNA-binding proteins to protease

Detergents n Are polar lipids, amphipathic u One hydrophobic portion (aliphatic or aromatic) F Soluble in lipids u One hydrophilic portion F So also soluble in aqueous environment n Intercalate into phospholipid bilayers of cell and solubilize... u membrane lipids and u membrane proteins

Detergents in our extract buffer n Tween - polyoxyethylene (20) sorbitol monolaurate or palmitate u good solubilization, mildly denaturing u non-ionic u aliphatic n Nonidet P40 - polyoxyethylene (9) p-t-octylphenol u good solubilization, weakly denaturing u non-ionic u aliphatic/aromatic

Proteinase K n Highly active enzyme that degrades proteins u Makes DNA accessible as a PCR template u pH for optimum activity is 7.8, but 8.3 is OK

Reaction temperature n o C u Proteinase K works best at o C u Higher temperature used to F Inhibit DNAses in the extract F Contribute to protein denaturation

Reaction time n One hour u Preliminary tests were done to establish that this time is long enough to make DNA accessible for PCR

Ending the reaction n Heat inactivate the proteinase K at 95 o C u If left active, Proteinase K can degrade the PCR polymerase in the next step F No polymerase, no product, no assay!