Transposon Products, Technical Appendix

Template Generation System™ II Kit, (TGS™ II Kit)

Specification of Kit Components

Entranceposon (CamR) and Entranceposon (KanR)
20 µl	20 ng/µl in TE buffer

The Entranceposons are composed of inverted repeats of bacteriophage Mu right end sequences, including the MuA binding sites R1 and R2, flanking the selectable marker gene, either cat (CamR) or npt (KanR). The Entranceposons provided in the kit are in precut configuration. Consequently, the first step in the Mu transposition, the donor cleavage, is by-passed in the reaction. This modification facilitates efficient transposition complex assembly and ensures sufficient stability of the complex.

MuA Transposase
20 µl	0.22 µg/µl in MuA Storage Buffer:
	HEPES, pH 7.6	25 mM
	EDTA	0.1 mM
	DTT	1 mM
	KCl	300 mM
	Glycerol	50 % (v/v)

A single purified polypeptide that catalyzes the in vitro transposition reaction. Isolated from an E. coli strain carrying the cloned MuA gene from bacteriophage Mu.

5x Reaction Buffer for MuA Transposase
100 µl	Component	5x conc.
	Tris-HCl, pH 8.0	125 mM
	MgCl2	50 mM
	NaCl	550 mM
	Triton® X-100	0.25 %
	Glycerol	50 % (v/v)

Control Target DNA
10 µl	370 ng/µl in TE buffer
	6.6 kb Hind III fragment of bacteriophage lambda DNA cloned into the Hind III site of the vector pUC19.

Primers for insertion mapping and sequencing:
pUC Fwd*	400 µl 25 µM in dd water
	5’-AGCTGGCGAAAGGGGGATGTG-3’
	Tm 73.5 °C*
Binding site in the vector pUC19 between the basepairs 307 and 327.
pUC Rev *	400 µl 25 µM in dd water
	5’-TTATGCTTCCGGCTCGTATGTTGTGT-3’
	Tm 71.6 °C
Binding site in the vector pUC19 between the basepairs 535 and 510.
Mu End **	800 µl 25 µM in dd water
	5’-GTTTTTCGTGCGCCGCTTCA-3’
	Tm 72.5 °C
Seq A ***	250 µl 10 µM in dd water
	5’-ATCAGCGGCCGCGATCC-3’
Seq B ****	250 µl 10 µM in dd water
	5’-TTATTCGGTCGAAAAGGATCC-3’

*Note: These primers anneal to the DNA flanking the polylinker of the pUC19 vector at opposite sides, with orientation towards the polylinker. The binding site sequences are well conserved in the plasmid “evolution”, and therefore the primers can be used with various vectors derived from the pUC vectors.

**Important: Binding site at each end of the Entranceposon. Do not use this primer for sequencing an insertion clone that contains a complete copy of the Entranceposon. However, use this primer if the PCR product from the colony-PCR reaction is sequenced directly.

***For sequencing outward from the left end of the Entranceposon. Binding site in both versions of the Entranceposon between basepairs 71 and 55.

****For sequencing outward from the right end of the Entranceposon. Binding site between basepairs 1245 and 1265 in the Entranceposon (CamR), and between basepairs 1138 and 1158 in the Entranceposon (KanR).

Mutation Generation System™ Kit, MGS™ Kit

Specification of Kit Components

Entranceposon (M1-CamR) and Entranceposon (M1-KanR)
10 µl	100 ng/µl in TE buffer

Entranceposon (M1-CamR) and (M1-KanR) are composed of inverted terminal repeats of modified bacteriophage Mu right end sequence with a NotI site, flanking the selectable marker gene, either cat (CamR) or npt (KanR). The marker genes code for resistance to the antibiotics chloramphenicol and kanamycin, respectively.

MuA Transposase
10 µl	0.22 µg/µl in MuA Storage Buffer
	HEPES, pH 7.6	25 mM
	EDTA	0.1 mM
	DTT	1 mM
	KCl	300 mM
	Glycerol	50 % (v/v)

A single purified polypeptide that catalyzes the in vitro transposition reaction. Isolated from an E. coli strain carrying the cloned MuA gene from bacteriophage Mu.

5x Reaction Buffer for MuA Transposase
100 µl	Component	5x conc.
	Tris-HCl, pH 8.0	125 mM
	MgCl2	50 mM
	NaCl	550 mM
	Triton X-100	0.25 %
	Glycerol	50 % (v/v)

Control Target DNA
10 µl	370 ng/µl in TE buffer
6.6 kb Hind III fragment of bacteriophage lambda DNA cloned into the Hind III site of the vector pUC19.

NotI Miniprimer
50 µl	25 µM in dd water
	5’-TGCGGCCGCA-3’
	Tm 57.5 ºC

The Miniprimer anneals to each DNA strand at the 15 bp insertion site generated by the Mutation Generation System Kit (MGS Kit)

Tm calculations were performed essentially as described by Breslauer et al. (1986) PNAS 83, 3746-3750.

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