Today's date:	9/19/05
PCR name:	S7A
Purpose:	Distinguish between Irs1 S307A mutant (S7A) knockin and control knockin (CK) alleles. Note: this is an allele-specific PCR; a wt (+) allele, gives same bands as CK allele. Digest is not required.
Samples:
1
2
3
4
5
			# of rxns
Reactions (ul):	X		11		Profile name:		JBM&S7A
DNA	1				step	time (min:sec)	temp (C)
H20	11.5		126.5		1	15:00	95
10X buffer	2		22		2	:30	94
4mM dNTPs	1.5		16.5		3	:20	64
10pmol/ul primers					4	:45	72
S7AF3	1		11		go to	step 2	34 times
NBR	1		11		5	10:00	72
BF	1		11		6	hold	4
S7AR (1010)	1		11
	20	ul	209	ul
+ HotStar Taq	0.18	ul	1.98
Primers:	Sequence: (5' to3')							Tm
S7AF3	GCAGCAAAAGCCAGTCTTCATCC							59.0
NBR (not BamHI)	AGGCACGCACCCGGAAGGAG							64.9
BF (BamHI)	AGTATGGTGGGTGGGAAACCAGGA							61.9
S7AR (1010)	CTGGGGTGACACTGCGGAAGG							62.7
Products:	from allele(s)		length (bp)	digests?		primers
common outer	all alleles		542	BamHI (S7A)*		S7AF3 + S7AR
spurious:	all alleles (occasionally)		~280			don't know origin of this band; disregard
S7A-specific (A)	S7A		399			BF + S7AR
wt-specific (S)	CK, +, etc		185			S7AF3 + NBR
				(* from the S7A allele only, the common band from outer
				primers can be digested with BamHI)
Genotypes:	bands
+/+	542 (variable), 185
CK/CK	542 (variable), 185
S7A/CK or S7A/+	542 (variable), 399, 185
S7A/S7A	542 (variable), 399