139pl120

EZ::TN™ <DHFR-1> Tnp Transposome™ Kit
Cat. No. TSM99D1
The EZ::TN™ <DHFR-1> Tnp Transposome™
EZ::TN™ <DHFR-1> Tnp Transposome™
is the stable complex formed between the EZ::TN Kit Contents
Transposase enzyme and the EZ::TN <DHFR-1>Transposon. The EZ::TN <DHFR-1> Transposon EZ::TN™ <DHFR-1> Tnp Transposome™ contains the dihydrofolate reductase (DHFR) gene (trimethoprim resistance) that is functional in E. coli, flanked by hyperactive 19 basepair Mosaic End (ME) EZ::TN Transpo-saserecognition sequences. The EZ::TN Trans-posome can be electroporated into living cells where the EZ::TN Transposase is activated by Mg2+ in the host's cellular environment resulting in randominsertion of the EZ::TN Transposon into the hostgenomic DNA.1-4 Quality Control: EZ::TN <DHFR-1> Tnp Transpo-
some activity is assayed by electroporation into a
Unlabeled forward and reverse transposon-spe- recA— E. coli host strain having a transformation cific primers are supplied in the kit. The primers efficiency of >109 cfu/µg DNA. Assays must yield can be used for bi-directional DNA sequencing or >106 trimethoprimR colonies/µg or >104 tri- mapping of transposon insertion sites in target methoprimR colonies/µl of transposome respec- genomic DNAs, including direct sequencing from tively. Primers are function-tested in a DNA cycle sequencing reaction using the SequiThermEXCEL™ II DNA Sequencing Kit and in a PCR Applications:
reaction using a plasmid containing an EZ::TN • Create gene "knockouts" by insertional muta- <DHFR-1> Transposon as template.
Contaminating Activity Assays: All components
• Sequence bacterial genomic DNA directly from of the EZ::TN <DHFR-1> Tnp Transposome Kit are transposon insertions, without cloning.
free of detectable DNase and RNase activities as • Insert priming sites for mapping or PCR into judged by agarose gel electrophoresis following over-digestion assays, with the exception of the • Insert a trimethoprim-resistance marker into inherent endonucleolytic function of the EZ::TN Related Products: The following products are
Product Specifications
Storage Temperature: Store only at -20oC in a
− TransforMax™ EC100™ Electrocompetent Storage Buffer: The EZ::TN <DHFR-1> Tnp
Transposome is supplied in a 50% glycerol solution containing 27.5 mM Tris-HCl (pH 7.5), X-100 and 0.5 mM dithiothreitol. The DHFR-1FP-1 Forward and DHFR-1 RP-1 Reverse Primers References:
are supplied in 10 mM Tris-HCl, (pH 7.5), 1 mM 1. Hoffman, L.M. et al., (1999) Current Genetics 35, 304.
2. Reznikoff, W. and Goryshin, I. Y., (1999) Epicentre Forum 6(2), 5.
Size: Reagents included in the kit are sufficient
3. Hoffman, L.M. and Jendrisak, J., (1999) for 10 in vivo transposon insertion reactions.
Epicentre Forum 6(3), 1.
4. Goryshin, I. Y. et al., (2000) Nat. Biotechnol. 18, 97.
EPICENTRE
EZ::TN™ <DHFR-1> Tnp Transposome™ Kit
Protocol
Electroporation of Host Cells with EZ::TN <DHFR-1> Tnp Transposome and
Selection of Transposition Clones:
Electroporate electrocompetent cells using 1 µl of the EZ::TN <DHFR-1> Tnp Transposome. Theelectrocompetent cells should have a transformation efficiency of >107 cfu/µg of DNA, but use cells ofthe highest transformation efficiency possible to maximize the number of transposon insertion clones.
Perform electroporation according to the equipment manufacturer's recommendations.
Immediately recover the electroporated cells after electroporation. Even slight delays in initiating the
cell recovery process will result in a reduced number of transposition clones. For E. coli, add SOC
medium to the electroporation cuvette to 1 ml final volume immediately after electroporation. Pipette the
medium/cells gently to mix. Transfer to a tube and incubate on a 37oC shaker for 30-60 minutes to
facilitate cell outgrowth.
Important Note: EZ::TN <DHFR-1> transposition clones are selected on trimethoprim plates. Tri-
methoprim is an antimetabolite, not an antibiotic, and the sensitivity of dihydrofolate reductase to this
compound varies widely among strains of bacteria, including E. coli. We have found E. coli strain
DH10B (Life Technologies, Inc.) to be sensitive to 10 µg/ml of trimethoprim. We recommend testing the
sensitivity of your strain by plating on media containing a range (10-200 µg/ml) of trimethoprim. Choose
a concentration of that results in no visible growth or a light haze of small, pin point colonies. Another
strain must be chosen if growth is confluent.
If working with E. coli, dilute aliquots of the recovered cells (e.g. 1:10 and 1:100). Plate 100 µl ofundiluted cells and each cell dilution separately on plates containing trimethoprim (a stock of 10 mg/mlcan be made in dimethylformamide). Store the unused portion of the electroporated cells at +4oC for upto 2 days in the event that additional plates need to be prepared. The number of trimethoprimRcolonies/µl of EZ::TN <DHFR-1> Tnp Transposome will be dependent on the transformation efficiency ofthe cells used and the level of expression of the DHFR gene in that species. EPICENTRE's TransforMaxEC100 Electrocompetent E. coli (available separately) have a transformation efficiency of >1 x 109cfu/µg and are ideal for this application.
Primer Information
DHFR-1 FP-1 Forward Primer
DHFR-1 RP-1 Reverse Primer
5' - GGCGGAAACATTGGATGCGG - 3'
5' - GACACTCTGTTATTACAAATCG - 3'
Length: 20 nucleotides
Length: 22 nucleotides
G+C content: 12
G+C content: 8
Molecular Weight: 6228 daltons
Molecular Weight: 6672 daltons
Temperatures of Dissociation & Melting:
Temperatures of Dissociation & Melting:
((81.5 + 16.6 (log [Na+])) +
((81.5 + 16.6 (log [Na+])) +
([41 (#G+C) - 500] / length) method)
([41 (#G+C) - 500] / length) method)
EPICENTRE
EZ::TN™ <DHFR-1> Tnp Transposome™ Kit
Triton is a registered trademark of Rohm & Haas, Philadelphia, Pennsylvania.
EZ::TN, Transposome, TransforMax, EC100, MasterPure and SequiTherm EXCEL are trademarks of EPICENTRE, Madison, Wisconsin.
EPICENTRE
EZ::TN™ <DHFR-1> Tnp Transposome™ Kit
Restriction Enzymes that cut the EZ::TN <DHFR-1> Transposon 1 to 3 times: Restriction Enzymes that cut the EZ::TN <DHFR-1> Transposon 4 or more times: Restriction Enzymes that do not cut the EZ::TN <DHFR-1> Transposon: EPICENTRE
EZ::TN™ <DHFR-1> Tnp Transposome™ Kit
EZ::TN™ <DHFR-1> Transposon 887 bp.
1 CTGTCTCTTA TACACATCTC AACCATCATC GATGAATTCG AGCTCGGTAC 51 CCGGATAGAC GGCATGCACG ATTTGTAATA ACAGAGTGTC TTGTATTTTT 101 AAAGAAAGTC TATTTAATAC AAGTGATTAT ATTAATTAAC GGTAAGCATC 151 AGCGGGTGAC AAAACGAGCA TGCTTACTAA TAAAATGTTA ACCTCTGAGG 201 AAGAATTGTG AAACTATCAC TAATGGTAGC TATATCGAAG AATGGAGTTA 251 TCGGGAATGG CCCTGATATT CCATGGAGTG CCAAAGGTGA ACAGCTCCTG 301 TTTAAAGCTA TTACCTATAA CCAATGGCTG TTGGTTGGAC GCAAGACTTT 351 TGAATCAATG GGAGCATTAC CCAACCGAAA GTATGCGGTC GTAACACGTT 401 CAAGTTTTAC ATCTGACAAT GAGGACGTAT TGATCTTTCC ATCAATTAAA 451 GATGCTTTAA CCAACCTAAA GAAAATAACG GATCATGTCA TTGTTTCAGG 501 TGGTGGGGAG ATATACAAAA GCCTGATCGA TCAAGTAGAT ACACTACATA 551 TATCTACAAT AGACATCGAG CCGGAAGGTG ATGTTTACTT TCCTGAAATC 601 CCCAGCAATT TTAGGCCAGT TTTTACCCAA GACTTCGCCT CTAACATAAA 651 TTATAGTTAC CAAATCTGGC AAAAGGGTTA ACAAGTGGCA GCAACGGATT 701 CGCAAACCTG TCACGCCTTT TGTGCCAAAA GCCGCGCCAG GTTTGCGATC 751 CGCTGTGCCA GGCGTTAGGC GTCATATGAA GATTTCGGTG ATCCCTGAGC 801 AGGTGGCGGA AACATTGGAT GCGGGGATCC TCTAGAGTCG ACCTGCAGGC 851 ATGCAAGCTT CAGGGTTGAG ATGTGTATAA GAGACAG The transposon sequence can be downloaded at the URL: www.epicentre.com/technical.htm.

Source: http://www.ebiotrade.com/buyf/productsf/epicentre/relation/139PL120.PDF