Table of Contents 1. Description...2 2. Kit Components...2 3. Storage...2 4. References...2 5. Principle...4 6. Protocol...5 7. Notes on Product...5 8. Note on Protocol...6 9. Related Products...6 Also available from TaKaRa DNA cloning / labeling systems DNA Ligation Kit Ver.1.0 (rapid ligation system)...#6021 DNA Ligation Kit Ver.2.1...#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 Deletion Kit for Kilo-Sequencing (generation of nested deletions)...#6030 DNA extraction cartridges SUPREC -01(100 cartridges) PCR * Related products Takara Taq Takara Ex Taq PCR Amplification Kit LA PCR Kit Ver.2 RNA PCR Kit Ver.3 RNA LA PCR Kit LA PCR in vitro Cloning Kit LA PCR in vitro Mutagenesis Kit (elution from agarose gels)...#9040...#r001... #RR001...#R011... #RR013... #RR019... #RR012... #RR015... #RR016 * The polymerase chain reaction (PCR) process is covered by patent owned by Hoffmann-La Roche. 1
1. 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 screening colonies. This novel system uses the pkf3 vector (chloramphenicol-resistant, Cm R ) containing the rpsl+ and the competent host strain TH2 (derived from HB101) which 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 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)... 20 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 39 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 (Cat.#9056) Content E.coli TH2 Competent Cells...100 μl 10 pbr322 DNA (0.1 ng/μl)... 10 μl SOC medium... 1 ml 10 Composition of SOC medium 2 % tryptone 0.5 % yeast extract 10 mm NaCl 2.5 mm KCl 10 mm MgSO4 10 mm MgCl2 20 mm Glucose 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 (rb -, mb - ), reca13, ara - 14, proa2, lacy1, galk2, rpsl20, xyl - 5, mtl - 1, leu B 6, thi - 1, trpr624 Transformation efficiency of TH2 cells 10 7 transformants/μg DNA when using 1 ng puc119 DNA. 3. Storage : pkf3 DNA : - 20 E.coli TH2 Competent Cells : - 80 (using dry ice/ethanol) 4. References : 1) Toba-Minowa, M. and Hashimoto-Gotoh. (1992) Gene 121, 25-33. 2) Hashimoto-Gotoh, T. et al. (1993) Gene 137, 211-216. 2
Fig. 1 Cloning site of pkf3 Fba I and Cla I are affected by dam methylase. Nco I-EcoT14 I-(Dsa I) (Spe I) PshB I (Spe I) Hpa I Pvu II Sac I-Ban II Nsp V Bal I-(Eae I) ApaL I 1.5 Xho I-(Ava I) Hgl E II 2.0 amber Sph I ori Nde I Mlu I Bst1107 I-(Acc I) EcoR I 0/2246 POtrp Aor51H I pkf3 2246 bp 1.0 Fsp I Spl I Hind III rpsl + 4am BstP I-EcoO 65 I Nhe I BspM I 0.5 Sca I Ssp I Sse8387 I-Pst I cat-5 Fba I Not I-Eco52 I-(Eae I) Bgl II Sma I-(Ava I) BamH I Kpn I Sac II-(Dsa I) Xba I Ava II Cla I Pvu I Stu I Acc III Sal I-(Acc I) pkf3 Primer F1 PshB I (Spe I) (Spe I) Hpa 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 Fsp I EcoR 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 (Dsa I) Kpn I Sac II Xba I Acc III Ava II (Acc I) Sal I Pvu I Cla I pkf3 Primer R2 Stu I Nhe I pkf3 Primer R1 Enzymes enclosed with ( ) cut pkf3 at several sites. As the enzymes marked with are affected by dam methylase, they do not cut pkf3 DNA. The supplied pkf3 DNA is methylated with dam methylase. 3
5. 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-), m a k i n g i t S t r e p t o m y c i n - resistant(sm R ). (A) Host genome E. coli (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. (B) Streptomycin-resistant + transform rpsl + rpsl + Plasmid Vector 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 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 plasmid (Cm resistance) with the foreign gene insert (Sm resistance). (C) Streptomycin-sensitive rpsl + + foreign gene to be inserted + transform Plasmid Vector Streptomycin-resistant 4
6. Protocol : 1. Digest 1 μg of pkf3 DNA with a restriction enzyme. 2. Collect the digested pkf3 DNA fragments through phenol/chloroform extraction followed by ethanol precipitation. 3. Add 35 ~ 350 fmol of insert DNA into 35 fmol (approx. 50 ng) of the collected DNA fragments. 4. Incubate at 16 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 (Becton Dickinson #352059 or #352057) 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 for 45 seconds. 8. Place the tube on ice for 1 ~ 2 min. 9. Add SOC medium prewarmed at 37 to the total volume of 1 ml. 10. Incubate at 37 with shaking (160 ~ 225 rpm) for an hour. 11. Spread an appropriate amount (50 ~ 100 μl) * of the reactant onto the L-broth plate containing 12 μg/ml Chloramphenicol and 50 μg/ml Streptomycin. 12. Incubate at 37 for overnight. * In case of a plate of 9 cm diameter, use in less than 100 μl per plate. 7. Notes on Product : 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 packed with dry ice/ethanol. 3. When transforming, microfuge can also be used instead of Falcon tube (Becton Dickinson 352059,352057), however, transformation efficiency in using microfuge 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 optimized when changing the reaction scale, ex. the amount of competent cells, or using a different tube. (For example, when using microfuge tube, incubate at 42 for 1 min.) 6. L-broth or ψ b-broth can be available instead of SOC medium, however, transformation efficiency may be lower. L-broth per liter Final concentration Bacto tryptone 10 g 1 % Bacto yeast extract 5 g 0.5 % NaCl 5 g 10 mm Adjust to ph7.5 with 1N NaOH. Autoclave. ψb-broth per liter Final concentration Bacto tryptone 20 g 2 % Bacto yeast extract 5 g 0.5 % MgSO4 7H2O 5 g 10 mm Adjust to ph7.5 with 1N KOH. Autoclave. 7. It is not recommended to freeze and store the thawed competent cells. However, if necessary, freeze in a dry ice/etoh bath and return to - 80. The transformation efficiency can be lowered by more than one magnitude. 8. When dilution of transformant is required, dilute with the medium used in '6. Protocol 9'. 5
8. 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 Aor13H I... #1224 Aor51H I... #1118 Ava II... #1008 Bal I... #1009 BamH I... #1010 Ban II... #1012 Bgl II... #1021 BstP I... #1025 BspT104 I... #1225 Bst1107 I... #1028 Cla I... #1034 EcoO65 I... #1135 EcoR I... #1040 EcoT14 I... #1038 Eco52 I... #1039 Fba I... #1045 Hind III... #1060 Hpa I... #1064 Kpn I... #1068 Mlu I... #1071 Nco I... #1160 Nde I... #1161 Not I... #1166 Nsb I... #1226 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 Sse8387 I... #1183 Stu I... #1088 VpaK11B I... #1196 Xba I... #1093 Xho I... #1094 9. Related Products : pkf3 DNA...#3100 E.coli TH2 Competent Cells...#9056 pkf3 Sequencing Primer F1...#3890 pkf3 Sequencing Primer R1...#3893 pkf3 Sequencing Primer R2...#3894 Note : This product is intended to be used for research purpose only. They are not to be used for drug or diagnostic purposes, nor are they intended for human use. They shall not to be used products as food, cosmetics, or utensils, etc. Takara products may not be resold or transfered, modified for resale or transfer, or used to manufacture commercial products without written approval from TAKARA BIO INC. If you require licenses for other use, please call at +81 77 543 7247 or contact from our website at www.takara-bio.com. 6 Phone: +81-77-543-7247 Fax: +81-77-543-9254