Today's date:	9/19/05
PCR name:	DG
Purpose:	Distinguish between Ptpn11 (ie Shp2) dominant active (D61G) mutant allele and wt (+) allele. This an allele specific PCR. Digest is not necessary.
Samples:
1
2
3
4
5
			# of rxns
Reactions (ul):	X		13		Profile name:		DG
DNA	1				step	time (min:sec)	temp (C)
H20	11.5		149.5		1	15:00	95
10X buffer	2		26		2	:30	94
4mM dNTPs	1.5		19.5		3	:20	62
10pmol/ul primers					4	:45	72
DOF	1		13		go to	step 2	34 times
DR1	1		13		5	10:00	72
GF1	1		13		6	hold	4
GOR	1		13
	20	ul	247	ul
+ HotStar Taq	0.18	ul	2.34
Primers:	Sequence: (5' to3')							Tm
DOF (D outer)	GCCAGCCATTCAAAGTAGAATCTC							56.0
DR1	CATAGAGGTCATAGTAGTCCCCA							54.8
GF1	CATCAAGATTCAGAACACCGGTGG							57.9
GOR (G outer)	ACCTGGTCACGAAGATGTCACTGA							59.8
Products:	from allele(s)		length (bp)	digests?		primers
common outer	all alleles		429	AgeI (DG)*		DOF + GOR
wt-specific (D)	+		270			DOF + DR1
mutant-specific (G)	DG		198			GF1 + GOR
				(* from the DG allele only, the common band from outer
				primers can be digested with AgeI.)
Genotypes:	bands
+/+	429 (variable), 270
DG/+	429 (variable), 270, 198
DG/DG	429 (variable), 198