Enforcement Cloning System pkf3 (Code No. 6086) E. coli TH2 Competent Cells (Code No. 9056) Table of Contents

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1 E. coli TH2 Competent Cells (Code No. 9056) v Table of Contents 1. Description Kit Component Storage References Principle Protocol Note on Product Note on Protocol Related Products...6 DNA cloning / sequencing / labeling systems DNA Ligation Kit Ver.1.0 (rapid ligation system)... #6021 DNA Ligation Kit Ver #6022 DNA Ligation Kit <Mighty Mix>... #6023 DNA Blunting Kit (blunting & ligation system)... #6025 MEGALABEL (DNA 5' labeling system)... #6070 Ladderman Labeling Kit (random primed labeling system)... #6046 Ladderman Dideoxy Sequencing Kit (for CTP labeling)... #6017 Ladderman Dideoxy Sequencing Kit (for ATP labeling)... #6018 Ladderman Dideoxy Sequencing Core Kit for Autosequencer... #6102 Takara Taq Cycle Sequencing Kit... #R014 Takara Taq Cycle Sequeincing Core Kit... #R018 Deletion Kit for Kilo-Sequencing (generation of nested deletions)... #6030 DNA extraction cartridges SUPREC -01(100 cartridges) (elution from agarose gels)... #9040 SUPREC -02 (100 cartridges) (purification of PCR products, concentration, buffer exchange)... #9041 PCR* Related products Takara Taq... #R001 Takara Ex Taq... #RR001 PCR Amplification Kit... #R011 LA PCR Kit Ver.2... #RR013 RNA PCR Kit Ver.3... #RR019 RNA LA PCR Kit... #RR012 LAPCR in vitro Cloning Kit... #RR015 LA PCR in vitro Mutagenesis Kit... #RR016 *The polymerase chain reaction (PCR) process is covered by patent owned by Hoffmann-La Roche. URL: TAKARA BIO INC. 1

2 E. coli TH2 Competent Cells (Code No. 9056) v Description: Enforcement Cloning System pkf3 (for 10 clonings) Cat.#6086 Takara's Enforcement Cloning System pkf3 has been developed to achieve simple and sensitive detection of recombinant DNA clones without the need for screening colonies. This novel system uses the pkf3 vector (chloramphenicol-resistant Cm R ) containing the rspl + and the competent host strain TH2 (derived from HB101) which is streptomycin-resistant (Sm R ). The foreign gene of interest is inserted into the multiple cloning site of the pkf3 vector and the TH2 cells are transformed and plated on LB medium containing Cm and Sm. The only colonies which will grow in the presence of these antibiotics are those which have obtained the plasmid (Cm resistance) with the foreign gene insert (Sm resistance). It can be used in various applications such as library, subcloning of PCR products, and colony hybridization. 2. Kit Components: 1. pkf3 DNA (0.5 µg/µl) µl Plasmid vector pkf3 DNA was constructed to allow enforcement cloning. It contains chloramphenicol-resistant gene originated from phsg399 and synthetic rpsl gene at the downstream of trp promotor/operator. On the synthetic rpsl gene, amber mutation and multicloning site having cleavage sites of 44 restriction enzymes are designed. Also 7 primers* are available to be compatible with the long multicloning site of pkf3. (* Refer to the Fig.1 for their sequences and to '9. Related product' for their Cat. numbers.) 2.E.coli TH2 Competent Cells Content E.coli TH2 Competent Cells µl X 10 Control puc119 DNA (0.1 ng/µl) µl TH2 strain is derived from HB101 which has been generally used in gene recombination experiments due to its stable genetic character. It has a mutation (trpr624) in trpr gene encoding trp repressor protein of HB101. Being rpsl -, supe -, trpr -, it is suitable for enforcement cloning using pkf3 DNA. Genotype supe44, hsds20(r B-, m B- ), reca13, ara - 14, proa2, lacy1, galk2, rpsl20,xyl - 5, mtl - 1, leub6, thi - 1, trpr624 Transformation efficiency of TH2 cells 10 7 transformants / µg DNA when using 1 ng puc119 DNA. 3. Storage: pkf3 DNA : E.coli TH2 Competent Cells: -20 C -80 C (using dry ice/ ethanol) 4. Reference: 1) Toba-Minowa, M. and Hashimoto-Gotoh. (1992) Gene 121, ) Hashimoto-Gotoh, T. et al. (1993) Gene 137, TAKARA BIO INC. URL:

3 E. coli TH2 Competent Cells (Code No. 9056) Fig. 1 Cloning site of pkf3 Nco IEcoT14 IDsaI Spe I PshB I Hpa I Spe I Pvu II SacIBanII Nsp V ApaLI 1.5 XhoIAvaI BalIEaeI Hgl EII 2.0 amber Sph I ori NdeI MluI Bst1107I-Acc EcoRI 0/2.246 POtrp Aor51HI pkf3 2246bp FspI v rpsl 4am + SplI HindIII BstPIEcoO 65I NheI BspMI 0.5 Sca I SspI Sse8387IPstI FbaI NotIEco52IEaeI BglII SmaI-Ava I BamHI KpnI SacIIDsaI XbaI AccIII AvaII SalI-AccI ClaI PvuI StuI cat Fba I and Cla I are affected by dam methylase. pkf3 Primer F1 PshB I (Spe I) Hpa I (Spe I) (Dsa I) EcoT14 I Nco I Pvu II Nsp V Ban II Sac I pkf3 Primer F2 (Eae I) Bal I (Ava I) Xho I Sph I Nde I Mlu I (Acc I) Bst1107 I EcoR I Fsp I Aor51H I Hind III Spl I BstP I EcoO65 I pkf3 Primer R4 Sse8387 I Pst I BspM I pkf3 Primer F3 Fba I (Eae I) Not I Eco52 I Bgl II (Ava I) Sma I BamH I pkf3 Primer R3 Kpn I (Dsa I) Sac II Xba I Acc III Ava II (Acc I) Sal I Pvu I Cla I pkf3 Primer R2 Stu I Nhe I URL: pkf3 Primer R1 are affected by dam methylase, they do not cut pkf3 DNA. The supplied pkf3 DNA is methylated with dam. TAKARA BIO INC. 3

4 E. coli TH2 Competent Cells (Code No. 9056) v Principle: (A) The rpsl gene encodes r-protein S12 which is one of the components of the ribosome. Streptomycin(Sm) inhibits protein synthesis by binding S12. The E.coli host strain TH2 has a mutation in the rpsl gene(rpsl - ), making it Streptomycinresistant(SmR). (B) Transforming TH2 competent cells with the pkf3 plasmid (rpsl + ) confers the Streptomycin sensitivity since the rpsl-mutation is recessive to the wild type gene. The pkf3 vector also carries the gene for Chloramphenicol resistance (Cm R ). (C) Inserting a foreign gene into the multiple cloning site (rpsl + ) of the pkf3 plasmid disrupts the production of S12 protein, thereby transforming the TH2 cells to Streptomycin-resistant. Utilizing the above principle, Enforcement Cloning System pkf3 has been developed to achieve simple and sensitive detection of recombinant DNA clones without the need for screening colonies. The pkf3 vector contains rpsl + gene and the competent cells (derived from HB101) is Streptomycinresistant (Sm R ). The foreign gene of interest is inserted into the multiple cloning site of the pkf3 vector and the TH2 cells are transformed and plated on LB medium containing Cm and Sm. The only colonies which will grow in the presence of these antibiotics are those which have obtained plasmid (Cm resistance) with the foreign gene insert (Sm resistance). 4 TAKARA BIO INC. URL:

5 E. coli TH2 Competent Cells (Code No. 9056) v Protocol 7. Notes on Product 1. Digest 1 ng of pkf3 DNA with a restriction enzyme. 2. Collect the digested pkf3 DNA fragments through phenol/chloroform extraction followed by ethanol precipitation. 3. Add fmol of insert DNA into 35 fmol(approx. 50 ng) of the collected DNA fragments. 4. Incubate at 16 C for 30 min. to perform ligation reaction. DNA Ligation Kit Ver.1.0 (Cat.#6021),Ver.2.1(Cat.#6022) or Mighty Mix (Cat.#6023) is useful for the ligation. 5. Take part of the reaction (<20 µl and <10 ng) into a Falcon tube containing 100 µl of E.coli TH2 Competent Cells melted on the ice just before use. Mix gently. 6. Place the tube on ice for 30 min. 7. Incubate at 42 C for 45 seconds. 8. Place the tube on ice for 1-2 min. 9. Add SOC medium prewarmed at 37 C to the total volume of 1 ml. 10. Incubate at 37 C with shaking (160~225 rpm) for an hour. 11. Spread an appropriate amount ( µl)* of the reactant onto the L-broth plate containing 12 µg/ml Chloramphenicol and 50 µg/ml Streptomycin. 12. Incubate at 37 C for overnight. * In case of a plate of 9 cm diameter, use in less than 100 µl per plate. 1. Competent cells need to be transported packed in dry ice/ethanol, strictly kept the product temperature. 2. Competent cells not for immediate use must be stored freezed at -80 C packed with dry ice/ethanol. 3. When transforming, Eppendorf tube can also be used instead of Falcon tube (Becton Dickinson ,352057), however, transformation efficiency in using Eppendorf tube may be lower. 4. When using 100 µl of Competent cells, DNA to be transformed should be prepared with high purity and the amount should be less than 10 ng to get high yield. 5. The conditions for transformation should be optimiezed when changing the reaction scale, ex. the amount of Competent cells, or using a different tube. (For example, when using Eppendorf tube, incubate at 42 C for 1 min.) 6. L-broth or φb-broth can be available instead of SOC medium, however, transformation efficiency may be lower. <Preparation of medium> SOC medium per liter Final concentration Bacto tryptone 20 g 2% Bacto yeast extract 5 g 0.5% NaCl 0.5 g 10 mm KCl 1.86 g 2.5 mm Autoclave and cool. Add 5 ml 2M MgCl 2, 5 ml 2M MgSO 4 (final conc. 10 mm each), 20 ml 1 M Glucose (final conc. 20 mm). All of these solutions should be filtered through a 0.22-micron filter. And then, filter the whole medium again through a 0.22-micron filter. L-broth per liter Final concentration Bacto tryptone 10 g 1% Bacto yeast extract 5 g 0.5% NaCl 5g 10 mm Adjust to ph7.5 with 1N NaOH. Autoclave. φb-broth per liter Final concentration Bacto tryptone 20 g 2% Bacto yeast extract 5 g 0.5% MgSO 4 7H 2 O 5g 10 mm Adjust to ph7.5 with 1N KOH. Autoclavre. 8. When dilution is required, dilute with the medium used in '6 Protocol 9'. URL: TAKARA BIO INC. 5

6 E. coli TH2 Competent Cells (Code No. 9056) v Note in protocol Special attention should be paid to eliminate contamination of exonuclease activity. It is important to use a restriction enzyme of high purity to cut plasmid pkf3. If exonuclease contamination is present, the rpsl gene may be truncated and functional S12 protein will not be expressed. Colonies formed become Streptomycin-resistant without foreign gene insertion. It results in high background to lower the insertion efficiency. * Takara offers a wide range of high-purity restriction enzymes guaranteed through the QC check employing this enforcement cloning system with pkf3. The followings are checked by this system. Acc III... #1113 Aor51H I... #1118 Ava II... #1008 Bal I... #1009 BamH I... #1010 Ban II... #1012 Bgl II... #1021 BstP I... #1025 Bst1107 I... #1028 Cla I... #1034 EcoO65 I... #1135 EcoR I... #1040 EcoT14 I... #1038 Eco52 I... #1039 Fba I... #1045 Hind III... #1060 Kpn I... #1068 Mlu I... #1071 Nco I... #1160 Nde I... #1161 Not I... #1166 Nsp V... #1175 PshB I... #1109 Pst I... #1073 Pvu I... #1075 Pvu II... #1076 Sac I... #1078 Sac II... #1079 Sal I... #1080 Sma I... #1085 Sph I... #1180 Spl I... #1087 Sse8387 I... #1183 Stu I... #1088 Xba I... #1093 Xho I... # Related products pkf3 DNA... #3100 E.coli TH2 Competent Cells... #9056 pkf3 Sequencing Primer F1... #3890 pkf3 Sequencing Primer F2... #3891 pkf3 Sequencing Primer F3... #3892 pkf3 Sequencing Primer R1... #3893 pkf3 Sequencing Primer R2... #3894 pkf3 Sequencing Primer R3... #3895 pkf3 Sequencing Primer R4... # TAKARA BIO INC. Phone: Fax: URL: