Strategy SSCP

Elektrophorese-Technik

fluorotechnics companies

		Strategy of SSCP-Electrophoresis

	General: For unknown gen-loci a certain step by step strategy of optimization for the SSCP-electrophoresis has shown to be optimal: First step: Searching for the optimal buffer-system. Second step: What is the optimal temperature? Third step: Determinating of the optimal gel-matrix. The samples (Heat-Shock-Protein-Gene, HSP-70) are kindly provided by:C. Schlingmann, DND-Riagnostik, Rostock, Germany.

Dez 17, 2008

First Step: Searching for the optimal buffer-system
ETC offers 3 different buffer-system with 3 different pH-values: 1. DNA-Disc-Buffer (pH = 8.5), 2. DNA-SSCP-Buffer (pH = 9.0), 3. DELECT-Buffer (ph 7.5)The samples are diluted normaly 1+2 with formamide, XylenCyanol is added. Because there are also fast running SSCP-bands the first run should be performed till this colour has reached the anode, not longer! The temperature is set to the medium value of 12.5 C. The double stranded band lies at 185 bp, that means at the anode.




	The “DNA-SSCP”-Buffer




		The “DNA-Disc”-Buffer




		The “DELECT”-Buffer


	Gel-matrix is the special CleanGel-SSCP (~10%T). Temp: 12.5 C. Running time 2h 20 min. Result: The DELECT--Buffer is the only system with sharply reproduced single-strands. Further experiments were done with that buffer!


	Second step: What is the optimal temperature?


		20 ^oC

	12.5 ^oC

	5 ^oC


	The run at 12.5 ^oC is already done, now a colder and a warmer value should be checked. Please note: 2h 20 min is the run at 12.5 ^oC, then the 5 ^oC-run is 30 min longer and the run at 20 ^oC is 20 min shorter. Result: When run at 5 ^oC the mutation bands shows best differences!


	Third Step: Determination of the optimal gel-matrix


	Normally a gel-matrix of around 10%T is optimal for the very slow migrating single DNA-strands (corresponding to 2kb double-strands). In this case we have a gene-locus which produces relatively quick SSCP-pattern (corresponding to 800bp double-strands)In this cases probably the 15%T matrix is more suitable. The running time is 30 min longer than run in 10%T.




	The CleanGel 15%T with the DELECT-buffer at 5 °C.


	Now all the mutants are shown clearly, allel 2 is now also visible!This result cannot be extrapolated to other gene-loci! In contrast to double-stranded DNA, the single-strands are individuals. They will behave different in these different condition. New gene loci must be tested singulary!


	ETCs offer for SSCP-optimizers: The SSCP-Test-Kit (ETC 1020-10)