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Microsoft word - nzymutagenesis kit

MB01202 (10 mutations plus competent cells) MB01204 (24 mutations plus competent cells) Description: NZYMutagenesis kit is designed to make point mutations and delete or insert single or multiple nucleotides in a DNA sequence. The system requires the provision of two synthetic oligonucleotide primers containing the desired mutation. Incorporation of the oligonucleotide primers with NZYDNAChange generates a mutated plasmid containing staggered nicks, which resists Dpn I digestion (as the synthetic DNA is not methylated). The resulting mutated plasmid is recovered through transformation of NZYStar competent cells. DNA isolated from dam- Escherichia coli strains, including JM101 and SCS110, is not a suitable template for the mutagenesis reaction. Storage: Store competent cells at -80 °C on receipt. Other kit components may be stored at -20 °C. NZYMutagenesis kit components are stable for at least six months when stored under the recommended *only provided in MB01202 and MB01204 kits. NZYTech, Lda. Estrada do Paço do Lumiar, Campus do Lumiar, Edifício E – R/C, 1649-038 Lisboa, Portugal Tel: +351 213643514 Fax:+351 217151168 Primer design: Primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid. Primers should be between 30 and 45 bases in length, with a melting temperature (Tm) of at least 78 °C. The desired mutation should be in the middle of the primer with approximately 10-15 bases of correct sequence on both sides. A minimum GC content of 40% is advisable and primers should terminate in one or more C or G bases. Primer purification (FPLC, PAGE or HPLC) is strongly 1. Synthesize and purify two complimentary oligonucleotides containing the desired mutation flanked by 2. On ice, in a sterile, nuclease-free microcentrifuge tube, prepare the following reaction: *To test the efficiency of the system use 2 μl of control plasmid and 2 × 1.5 μl of each one of the 3. Gently mix and centrifuge the reactions in a microcentrifuge for 5 seconds. If using a thermal cycler without a heated lid, overlay the reaction mix with 1-2 drops of mineral oil to prevent evaporation 4. Proceed with the amplification following the cycling parameters outlined in Table 1. 5. Place reaction tubes on ice for 2 minutes. If desired, you may check the efficiency of the amplification by analysing 10 μl of the reaction on a 0.7-1% agarose gel. Proceed with the Dpn I digestion even if a 6. Add 1 μl of Dpn I directly in to the reaction (below the mineral oil if used). Gently mix, spin down the reaction and incubate at 37 °C for 1 hour to digest the non-mutated template DNA. NZYTech, Lda. Estrada do Paço do Lumiar, Campus do Lumiar, Edifício E – R/C, 1649-038 Lisboa, Portugal Tel: +351 213643514 Fax:+351 217151168 Table 1 – Cycling parameters for the NZYMutagenesis method Note: control plasmid is 6 kb in size and, therefore, use a 9 minute elongation period for the control 7. Transfer 5 µl of the Dpn I treated DNA to 100 µl of the ultracompetent cells. NZYStar cells are resistant to tetracycline. If the mutagenized plasmid contains only a tetR resistance marker, an alternative tetracycline-sensitive strain of competent cells must be used. 8. To determine the transformation efficiency, add 1 µl (10 ng) pUC19 control DNA to one tube containing 100 µl competent cells. Gently tap tube to mix. Do not mix cells by pipetting. 9. Incubate transformation reaction for 30 min on ice. 10. Heat shock cells at 42 °C for exactly 40 seconds. 12. Add 900 µl of pre-warmed SOC medium (not provided). 13. Shake the tubes at 200 rpm at 37 °C for 1 hour. 14. Centrifuge at 5000 rpm for 1 min. Remove 900 µl of supernatant. 15. Re-suspend cells by gently pipetting. Plate 100 µl of cells onto LB agar plates containing 100 µg/µl 16. For pUC19 control transformation directly plate 100 µl without spinning. 17. Incubate inverted plates overnight at 37 °C. endA1 hsdR17(r -, m +) supE44 thi -1 recA1 gyrA96 relA1 lac[F´ proA+B+ lacIqZ∆M15 :Tn10(TcR)] NZYTech, Lda. Estrada do Paço do Lumiar, Campus do Lumiar, Edifício E – R/C, 1649-038 Lisboa, Portugal Tel: +351 213643514 Fax:+351 217151168 1. PCR efficacy may be improved by increasing the amount of template DNA used (to a maximum of 100 ng of plasmid DNA per reaction). In this circumstance increase the incubation time with Dpn I to 2 hours. 2. The levels of dNTP may affect the efficiency of the PCR reaction. You may proceed to an optimization by varying the levels of dNTP in the reaction from 0,5 to 2 µl. 3. The amount of reaction used for transformation may be increased to a maximum of 10 µl to 100 µl of cells. 4. Avoid multiple freeze-thaw cycles for the dNTP mix. Thaw the dNTP mix once and prepare single 5. False priming and the formation of secondary structures may affect the mutagenesis reaction. Increasing the annealing temperature up to 68 °C may help improving the efficacy of the PCR reaction. NZYTech, Lda. Estrada do Paço do Lumiar, Campus do Lumiar, Edifício E – R/C, 1649-038 Lisboa, Portugal Tel: +351 213643514 Fax:+351 217151168



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