DI PCR Protocol

EGFP Genotyping Protocol at Donor's Laboratory for GENSAT BAC transgenic strains

Reagents:

Reagent / Constituent	Concentration	Volume (µL)
buffer (Qiagen)	10X	2.0
MgCl₂ (Qiagen)	15 mM	0.8
dNTPs (Qiagen)	2.5mM	1.6
Primer 1 Name: EGFP forward primer	50 µM	0.2
Primer 2 Name: EGFP reverse primer	50 µM	0.2
Primer 3 Name: Actin PCR control/forward	50 µM	0.4
Primer 4 Name: Actin PCR control/reverse	50 µM	0.4
Tag Polymerase (Qiagen)		0.2
Q solution (Qiagen)		4.0
Water		9.2
tail genomic DNA sample		1.0
Total Volume of Reaction:		20.0 µL

Primers:

#	Primer Name	Nucleotide Sequence (5' - 3')
1	EGFP forward primer	CCT ACG GCG TGC AGT GCT TCA GC
2	EGFP reverse primer	CGG CGA GCT GCA CGC TGC GTC CTC
3	Actin PCR control/forward primer	GAT GAC GAT ATC GCT GCG CTG GTC G
4	Actin PCR control/reverse primer	GCC TGT GGT ACG ACC AGA GGC ATA CAG

Cycles:

Step	Temp. (°C)	Delta per cycle	Time(secs.)	# of Cycles
1. Initial melting	94	-	180	1
2. Denaturization	94	-	30	-
3. Anealing	60	-	45	-
4. Elongation	72	-	45	-
Repeat steps 2, 3, 4			total cycles: 30
5. Amplification	72	-	600	1
Finish	4	-	indefinite	n/a

Electrophoresis:

2.5% Agarose gel at 200-250 mVolts for 30-45 mins.

Band #	Position	Genotype
1	~300 bp	EGFP
2	1 kb	Actin (control)

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Last Modified: April 20, 2005