Today's date:	9/19/05
PCR name:	A* (A star)
Purpose:	Distinguishes between wt (+) allele and Irs1 knockin (ki) mutant alleles FCT and CK when there is a possibility that these are present together. Digest of the PCR with BglII reveals whether knockin allele is FCT or control CK.
Samples:
1
2
3
4
5
		# of rxns
Reactions (ul):	X	11			Profile name:		IRS1KIGT
DNA	1				step	time (min:sec)	temp (C)
H20	13.5		148.5		1	15:00	95
10X buffer	2		22		2	:30	94
4mM dNTPs	1.5		16.5		3	:20	62
10pmol/ul primers					4	:45	72
cdsN2	1		11		go to	step 2	34 times
UTRrev1	1		11		5	10:00	72
					6	hold	4
	20	ul	209	ul
+ HotStar Taq	0.18	ul	1.98	ul
Primers:	Sequence: (5' to3')							Tm
cdsN2	GCAGTGAGGATGTAAAACGCCACAGCTCTGCATC							66.0
UTRrev1	AGAGAGAAGCCCTTCTGTGGCTGCTCCAAACACA							67.5
Products:	from allele(s)		length (bp)	digests?		primers
+	wt (+)		584	583		cdsN2 + UTRrev1
ki	ki (CK, S7A, JBM, etc)		649			cdsN2 + UTRrev1
ki	ki (FCT)		649	Bgl II		cdsN2 + UTRrev1
				(add 10 units BglII directly to 20 ul reaction)
Genotype:	bands (after digestion)
+/+	584 only
CK/+, S7A/+, etc	649 and 584
FCT/+	584, 509 and 140 (faint)			for FCT ki only, 649bp band digests with BglII
CK/CK	649
FCT/CK	649, 509 and 140 (faint)			for FCT ki only, 649bp band digests with BglII
FCT/FCT	509 and 140 (faint) only			for FCT ki only, 649bp band digests with BglII
					for FCT ki only, 649bp band digests with BglII