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From 2012.igem.org

I. Ordered Cross-over PCR Primers from Keck

• Primers were ordered with the name/sequence in the following table:

Name	Tm (Tm overhang/Tm anneal)	Length (bp)	Sequence (overCUTSITEanneal)
cT5_pAf	66.64 (25.23/58.79)	30 (10/20)	5'-tgacTCTAGAtttgctttgtgagcggataa-3' (XbaI)
cT5_pAr	69.08 (30.28/55.61)	25 (10/15)	5'-gtaaCTGCAGgagagcgttcaccga-3' (PstI)

• See Project Page for more info

II. Resuspend Lyophilized Primers for Cross-over PCR

• Spin down tubes
• Add the volume specified in the table of TE Buffer to the primer tubes

Name	Volume (uL)
p1f	208.35
p1r	217.78
p2f	199.75
p2r	199.6
p3f	199.75
p3r	190.55
p4f	248.1
p4r	149.85
p5f	127.75
p5r	199.65

• Vortex all tubes briefly
• Spin all tubes down
• Incubate on bench for 10 minutes
• Store at -20 ^oC