Gateway Enzymes

Frame:	ACA AGT TTG TAC AAA AAA GCA GGC T------AC CCA GCT TTC TTG TAC AAA GTG GT
Entry clone	N75-ACAAGTTTGTACAAAAAAGCAGGCT------------------ACCCAGCTTTCTTGTACAAAGTGGT-N75 N75-TGTTCAAACATGTTTTTTCGTCCGA------------------TGGGTCGAAAGAACATGTTTCACCA-N75 [-----------attL1-----------]        ORF       [-----------attL2-----------]
	X
Destination vector	ACAAGTTTGTACAAAAAAGCAGGCT-N100--------N100-ACCCAGCTTTCTTGTACAAAGTGGT TGTTCAAACATGTTTTTTCGTCCGA-N100--------N100-TGGGTCGAAAGAACATGTTTCACCA [-----------attR1------------]  ccdB  [-----------attR2------------]
	=
Expression clone	ACAAGTTTGTACAAAAAAGCAGGCT------------------ACCCAGCTTTCTTGTACAAAGTGGT TGTTCAAACATGTTTTTTCGTCCGA------------------TGGGTCGAAAGAACATGTTTCACCA [--------attB1----------]        ORF       [--------attB2----------]
	+
By- product	N75-ACAAGTTTGTACAAAAAAGCAGGCT-N100--------N100-ACCCAGCTTTCTTGTACAAAGTGGT-N75 N75-TGTTCAAACATGTTTTTTCGTCCGA-N100--------N100-TGGGTCGAAAGAACATGTTTCACCA-N75 [-------------attP1--------------]  ccdB  [-------------attP2--------------]

LR reaction

1. Assemble the reaction:

		5 µl	10 µl
	Entry clone (50-150ng)	x µl	x µl
	Destination vector	75 ng	150 ng
	5X LR Buffer	1 µl	2 µl
	TE pH 8.0	to 4 µl	to 8 µl
	LR Clonase	0.5µl	1 µl

1. Incubate at 25°C for 1 hour to overnight.
2. Transform into competent cells and plate on plates containing a suitable antibiotic.

original BP buffer (5x)

• 125 mM Tris HCl pH 7.5
• 25 mM EDTA
• 110 mM NaCl
• 25 mM Spermidine-HCl
• 5 mg/ml BSA

Note: BSA should be NON-acetylated, as Int is rapidly inactivated (at least in its pure form) by acetylated BSA

BP3 buffer (5x) - only works with attB1.1 and attB2.1

• 100 mM Tris pH 7.5
• 20 mM EDTA
• 225 mM NaCl
• 30 mM Spermidine-HCl
• 25% Glycerol

Original LR buffer (5x)

• 250 mM of Tris HCl pH 7.5
• 2.25 mM of EDTA
• 250 mM of NaCl
• 12.5 mM of spermidine HCl
• 1 mg/mL of BSA

Note: BSA should be NON-acetylated, as Int is rapidly inactivated (at least in its pure form) by acetylated BSA

LR4 buffer (5x) - for use with attL1.1 and attL2.1

• 200 mM Tris-Cl, pH 7.5
• 10 mM EDTA
• 320 mM NaCl
• 35 mM spermidine-HCl
• 25% Glycerol

Plasmids:

pMCSG13-IHF, Clonesdev 405

pET28b-Int, 406

pET28b-Xis, 407

Patents 5,888,732, 6,143,557, 6,171,861, 6,270, 969, and 6,277,608