Today's date:	9/19/05
PCR name:	ix1
Purpose:	Genotype intercross progeny (ix) simultaneously for three Irs1 alleles: wt (+) allele, Irs1 knockout (ko) allele, Irs1 knockin mutant alleles (ki) with neo (n) cassette at 3' end of coding region (examples CKn, FCTn)
Samples:
1
2
3
4
5
			# of rxns
Reactions (ul):	X		13		Profile name:		ix1
DNA	1				step	time (min:sec)	temp (C)
H20	12.3		159.9		1	15:00	95
10X buffer	2		26		2	:30	94
4mM dNTPs	1.5		19.5		3	:20	60
10pmol/ul primers					4	:45	72
cdsN2	0.8		10.4		go to	step 2	36 times
E	0.8		10.4		5	10:00	72
Irs1 neoF2	0.8		10.4		6	hold	4
Irs1 lower	0.8		10.4
	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
Irs1 neoF2	GCATCGCCTTCTATCGCCTTCTTG							60.0
Irs1 lower	TGGCCGCTCCCGAATTCAAT (has bad 3' end)							59.8
Products:	from allele(s)		length (bp)	digests?		primers
	ko		680			Irs1 neoR2 + Irs1 lower
spurious:	FCTn, CKn, etc		~650			don't know origin of this band; disregard
	CKn, FCTn, etc		387			cdsN2 + E
				(to distinguish CKn and FCTn, do PCR A with BglII digest)
Genotypes:	bands		Note:	if necessary, infer genotypes from parentage or…
+/+	none			confirm with PCR A
FCT/+, CK/+	387 only			distinguish with PCR A digested with BglII
ko/+	680 only			confirm with PCR Irs1
FCT/FCT, CK/CK	387 only			distinguish with PCR A digested with BglII
FCT/ko, CK/ko	680,  650 (faint), and 387			distinguish with PCR A digested with BglII
ko/ko	680 only			confirm with PCR Irs1