Today's date:	9/19/05
PCR name:	A
Purpose:	Distinguishes between wt (+) allele and Irs1 knockin mutant alleles (ki) still containing neo (n) cassette at 3' end of coding region (examples: CKn, FCTn, S7An, JBMn). Digest of this PCR with BglII is  also useful for distinguishing between CKn and FCTn alleles.
Samples:
1
2
3
4
5
		# of rxns
Reactions (ul):	X	13			Profile name:		IRS1KIGT
DNA	1				step	time (min:sec)	temp (C)
H20	13		169		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
cdsN2	1		13		go to	step 2	34 times
E	0.5		6.5		5	10:00	72
UTRrev1	1		13		6	hold	4
	20	ul	247	ul
+ HotStar Taq	0.18	ul	2.34	ul
Primers:	Sequence: (5' to3')							Tm
cdsN2	GCAGTGAGGATGTAAAACGCCACAGCTCTGCATC							66.0
E	GGGGCCCTCGACATAACTTCGTATAGCATACAT							63.3
UTRrev1	AGAGAGAAGCCCTTCTGTGGCTGCTCCAAACACA							67.5
Products:	from allele(s)		length (bp)	digests?		primers
+	wt (+)		584	no		cdsN2 + UTRrev1
ki	ki (CKn, S7An, etc)		387	no		cdsN2 + E
ki	ki (FCTn)		387	yes, Bgl II		cdsN2 + E
				(can add 10 units Bgl II directly to 20 ul reaction)
Genotype:	bands
+/+	584 only
CKn/+,S7An/+, etc	584 and 387
FCTn/+	584 and 387
FCTn/CKn, etc	584 only
	bands	after OPTIONAL digest with Bgl II (useful as compliment to PCR ix)
CKn/+	584, 387
CKn/ko	584, 387			ie ko gives same 584 bp band as + allele
FCTn/+	584, 247 and 140			for FCT ki only: 387 bp digests into 140 bp + 247 bp
FCTn/CKn	399, 247 and 140			for FCT ki only: 387 bp digests into 140 bp + 247 bp