Microarray Probe Preparation

Last updated: December 2, 2003 by Siew-Loon Ooi

Reagents:

5 µM (each) Cy3 UPTAGMix: U1=JB3323; Cy3-JB3327
5 µM (each) Cy5 UPTAG Mix: U1=JB3323; Cy5-JB3328
5 µM (each) Cy3 DOWNTAG Mix: D1=JB3324; Cy3-JB3329
5 µM (each) Cy5 DOWNTAG Mix: D1= JB3324; Cy5-JB3330

Primer Sequences

D1 = JB3324 = CGGTGTCGGTCTCGTAG

D2 = JB3326 = ATCGATGAATTCGAGCTCG

U1 = JB3323 = GATGTCCACGAGGTCTCT

U2 = JB3325 = CGTACGCTGCAGGTCGAC

JB3327 = 5’-Cy3 -GTCGACCTGCAGCGTACG

JB3328 = 5’-Cy5 -GTCGACCTGCAGCGTACG

JB3329 = 5’-Cy3 -CGAGCTCGAATTCATCGAT

JB3330 = 5’-Cy5 –CGAGCTCGAATTCATCGAT

Protocols

PCR Reaction:

	Volume;
Water	38
Takara Ex Taq Premix	50
5 µM Primer Mix	10;
Genomic DNA 50 ng/µl	2 µl
Total Volume	100;

Assemble PCR reactions as described above. For each genomic DNA template, the 100 µl reaction is aliquoted into two 50 µl aliquots to perform two independent PCR reactions.

PCR conditions are:

94遨 3'
{94遨 30" -> 50遨 30" -> 72遨 30"} 30X
72遨 5' -> 4遨

Comments
Cy3 and Cy5 dyes are light sensitive, try to minimize exposure to light by using aluminum foil to protect samples from light.
PCR : We have used both Platinum Taq Supermix (Invitrogen, Cat # : 11306016) and Takara Ex Taq Premix (Panvera, Cat #:RR003A) to perform PCR.
Genomic DNA Template concentrations: We have used 20 to 100 ng/µl of genomic DNA as template.