DD-PCR Problems Galore

Y4OLT at TTACS1.TTU.EDU Y4OLT at TTACS1.TTU.EDU
Sun Feb 12 02:38:16 EST 1995


I have been having some trouble getting anything close to what might be called
reproduceable results with my differential display products.  The protocol I've
been using is as follows:
		1.0 ul 1st strand cDNA
		2.5 ul 10X Taq buffer
		2.5 ul MgCl2
		2.5 ul dNTP's (final conc. 20uM)
		0.6 ul oligo dT (final conc. 0.5 uM)
		0.5 ul random primer(final conc. 0.5 uM)
		13.8 ul water
		1.2 ul 35-S dATP (1355Ci/mmol, 9.2 uM)
		0.4 ul Stoffel Fragment Taq polymerase
			(total volume 25 ul)
		PCR condit:94 for 30 sec
			   42 for 1 min
			   72 for 30 sec
			   Final soak at 72 for 5 min.

I've been running the products out on 6% sequencing gels.
My problems have ranged from completely blank gels to dark smears.
I've tried all of the usual methods to cure PCR problems (titrating MgCl2, DNA,
enzyme, etc.) and have had little luck.
Any suggestions would be greatly appreciated!!!!!!

Owatha Tatum
Dept. of Biological Sciences
Texas Tech University
MS 3131
Lubbock, TX  79409




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