Table of contents. I. Description II. Kit Components III. Reagents and Instruments Required IV. Storage V. Protocols...

Transcription

1 Table of contents I. Description... 2 II. Kit Components... 2 III. Reagents and Instruments Required... 2 IV. Storage... 2 V. Protocols... 3 VI-1. Procedure... 3 VI-2. Note... 4 VI. Control experiment... 5 VII. Cloning example of fragments with different lengths... 6 VIII. Comparison among PCR enzymes... 7 IX. Related products

2 I. Description : When PCR products are cloned into blunt-end vectors, the removal of extra bases at 3 -end and phosphorylation of 5 end are necessary depending on an used polymerases. Takara s is designed to allow this series of reaction in a simple method and quickly. As PCR products are blunt-ended in parallel with phosphorized at 5 -end, DNA fragments can be prepared into a ready-for-ligation just in a single reaction. Pretreatment of PCR products is not necessary, such as inactivation of enzymes, removal of unused dntps and primers, etc. This kit utilizes completely the same reagent that are included in Takara s efficient DNA Ligation Kit Ver. 2.1 (Cat. #6022), which achieves the series of operation efficiently and in a short time. II. Kit Components (24 reactions) : Blunting Kination Buffer 50 μl 2. Blunting Kination Enzyme Mix 25 μl 3. Ligation Solution I * μl 4. Control Vector (puc118-hinc II/BAP) 50 ng/μl 5 μl 5. Control Insert * ng/μl 10 μl 6. ddh2o 500 μl * 1 : Ligation Solution I is the same reagent that is included in DNA Ligation Kit Ver.2.1 (Cat. #6022). * 2 : 500 bp PCR fragment amplified with TaKaRa Taq by using λ-dna as a template. III. Reagents and Instruments Required but Not Supplied in the Kit : Dephosphorylated blunt-end vector Competent cell or Electro cell SOC culture medium LB plate IV. Storage : -20 2

3 V. Protocol : V-1. Procedure A. When PCR reactant is directly used : Blunting Kination reaction 1. Prepare the following reaction mixture in a micro tube. PCR reactant 10 Blunting Kination Buffer Blunting Kination Enzyme Mix ddh2o 15 μl 20 μl 2. Incubate at 37 for 10 min. 3. Mix 80 μl of distilled water and 100 μl of phenol/chloroform/isoamyl alcohol (25 : 24 : 1) together. 4. Centrifuge at 12,000 rpm for 5 min. at room temperature, then transfer the upper layer to a new tube. 5. Add equal amount of chloroform/isoamyl alcohol (24 : 1) and mix. 6. Centrifuge at 12,000 rpm for 5 min. at room temperature, then transfer upper layer to new tube. 7. Add 10 μl of 3M sodium acetate and 250 μl of chilled ethanol, and then mix. Keep it for 20 min. at Centrifuge at 12,000 rpm for 10 min. at 4. Remove the supernatant. 9. Wash the precipitate with chilled 70% ethanol. Centrifuge at 12,000 rpm for 5 min. at 4 and remove the supernatant. 10. Dry the precipitate. 11. Dissolve the precipitate in a suitable amount of TE water. Ligation reaction 12. Put appropriate volume of DNA solution obtained at step 5 into a new micro tube. 13. Add of dephosphorylated blunt-end vector ( ng/μl), and mix. 14. Add the equivalent volume of Ligation Solution I and mix. 15. Incubate at 16 for 1 hour. 16. Perform a transformation using the whole solution prepared at step 9. for 100 μl of competent cell. When carrying out a transformation by the electroporation method, exchange the buffer by ethanol precipitation before transformation. * When other bands than the target band appear in the PCR product, or when the shorter insert DNAs are exclusively cloned, please purity the target fragment by agarose gel electrophoresis. * Appropriate volume of PCR reactant is. When large volume is used, the reaction efficiency may lower. Please exchange the buffer by ethanol precipitation if necessary. B. When purified DNA fragment is used 3

4 Blunting Kination reaction 1. Prepare the following reaction mixture in a micro tube. DNA Fragment 10 Blunting Kination Buffer Blunting Kination Enzyme Mix ddh2o pmol X μl 20 μl 2. Incubate at 37 for 10 min. 3. Mix 80 μl of distilled water and 100 μl of phenol/chloroform/isoamyl alcohol (25 : 24 : 1) together. 4. Centrifuge at 12,000 rpm for 5 min. at room temperature, then transfer the upper layer to a new tube. 5. Add equal amount of chloroform/isoamyl alcohol (24 : 1) and mix. 6. Centrifuge at 12,000 rpm for 5 min. at room temperature, then transfer upper layer to new tube. 7. Add 10 μl of 3M sodium acetate and 250 μl of chilled ethanol, and then mix. Keep it for 20 min. at Centrifuge at 12,000 rpm for 10 min. at 4. Remove the supernatant. 9. Wash the precipitate with chilled 70% ethanol. Centrifuge at 12,000 rpm for 5 min. at 4 and remove the supernatant. 10. Dry the precipitate. 11. Dissolve the precipitate in a suitable amount of TE water. Ligation reaction 12. Put appropriate volume of DNA solution obtained at step 5 into a new micro tube. 13. Add of dephosphorylated blunt-end vector ( ng/μl), and mix. 14. Add the equivalent volume of Ligation Solution I and mix. 15. Incubate at 16 for 1 hour. 16. Perform a transformation using the whole solution prepared at step 9 for 100 μl of competent cell. When carrying out a transformation by the electroporation method, exchange the buffer by ethanol precipitation before transformation. * Appropriate insert/vector ratio (molar ratio) is 2:1-10:1. V-2. Note 1. Thaw Ligation Solution I on ice or in ice water. Mix well before use. Ligation Solution I would not be inactivated by freeze-thaw cycles. 2. When ligation efficiency is low, the following remedies are recommended : - Extend the reaction time to overnight - Add NaCl at the final concentration of 500 mm into the solution after ligation reaction, and perform transformation. Addition of salt increases the transformation efficiency. When all the above remedies would not improve the reaction, repurification of DNA is recommended. 3. Light blue colonies may appear on color selection plate, when short DNA fragment is cloned with a vector having lacz. This is because stop codon does not appear or frame shift does not occur, even after the insertion of on insert DNA. 4. When cloning short DNA fragments, inserts may be ligated among themselves and clone containing the linkage of several inserts may appear. 5. When mineral oil is used in PCR reaction, pay attention not to contaminate the reaction solution with mineral oil. 4

5 VI. Control experiment : 1. Prepare the following reaction mixture. Control Insert 10 Blunting Kination Buffer Blunting Kination Enzyme Mix ddh2o 15 μl 20 μl 2. Incubate at 37 for 10 min. 3. Mix 80 μl of distilled water and 100 μl of phenol/chloroform/isoamyl alcohol (25 : 24 : 1) together. 4. Centrifuge at 12,000 rpm for 5 min. at room temperature, then transfer the upper layer to a new tube. 5. Add equal amount of chloroform/isoamyl alcohol (24 : 1) and mix. 6. Centrifuge at 12,000 rpm for 5 min. at room temperature, then transfer upper layer to new tube. 7. Add 10 μl of 3M sodium acetate and 250 μl of chilled ethanol, and then mix. Keep it for 20 min. at Centrifuge at 12,000 rpm for 10 min. at 4. Remove the supernatant. 9. Wash the precipitate with chilled 70% ethanol. Centrifuge at 12,000 rpm for 5 min. at 4 and remove the supernatant. 10. Dry the precipitate. 11. Dissolve the precipitate in 20 μl of TE Buffer. 12. Prepare the following reaction mixture using the obtained filtrate. DNA Solution Control Vector Ligation Solution I 5 μl 6 μl Incubate at 16 for 1 hr. 14. Perform transformation of the E. coli JM109 Competent Cells (100 μl) using the whole volume. 15. Spread it on the plate containing Ampicilin, X-Gal, and IPTG. When a competent cell having the transformation efficiency of colonies/μg puc118 DNA is used, approximately colonies would be obtained as white colony per 50 ng vector. The sequence of both ends of Control Insert is 5 -GAC...GTC-3. When the protocol is performed in a correct method, Hinc II sites are regenerated and insert can be excised. 5

6 VII. Cloning example of fragments with different lengths : Five PCR products with different length (200 bp, 500 bp, 1,000 bp, 1,500 bp, and 2,000 bp) were amplified using Takara Taq and lambda DNA as a template. Following the protocol, they were blunt-ended, phosphorylated, and cloned into Hinc II site of dephosphorylated puc118. Then transformation was performed with E. coli JM109 Competent Cells, and color selection was done. Along with it, three PCR products with 1,000 bp, 1,500 bp, and 2,000 bp were purified from gel after agarose gel electrophoresis, and then they were applied to the same cloning experiment almost in the same volume as above, following the same protocol. For DNA recovery from gel, SUPREC -01 (Cat. #9040) was used. [Result] Insertion DNA Chain length (bp) White colony/blue colony (/50 ng puc118 DNA) Insert DNA/White colony / / / /10 1, / /10 1, / /10 2, / /10 A. When PCR products are directly used Insert DNA Chain length (bp) White colony/blue colony (/50 ng puc118 DNA) Insert DNA/White colony 1, / /10 1, / /10 2, / /10 B. When DNA fragments purified from agarose gel is used The competent cell used in this experiment had the transformation efficiency of colonies/μg puc 118 DNA. The presence of insert was verified through PCR. The results shows that gel purification helps to increase transformation efficiency. 6

7 VIII. Comparison among PCR enzymes : The 1,000 bp of PCR products were prepared with TaKaRa Taq, TaKaRa Ex Taq, TaKaRa LA Taq, and Pyrobest DNA polymerase * using lambda DNA as a template. Following the protocol of this kit, they were cloned into Hinc II site of puc118. Then the transformation was done using E. coli JM109 and then color selection was performed. DNA Polymerase used for PCR White colony/blue colony (/50 ng puc118 DNA) Insert DNA/White colony TaKaRa Taq / /6 TaKaRa Ex Taq / /6 TAKaRa LA Taq / /6 Pyrobest DNA polymerase / /6 The competent cell used in this experiment had the transformation efficiency of colonies/μg puc 118 DNA. The presence of insert was verified through PCR. The results shows that gel purification helps to increase transformation efficiency. * : Pyrobest DNA polymerase is not available in the U.S.A. IX. Related products : E. coli JM109 Competent Cells (Cat. #9052) * Competent Cells and Electro-Cells are not availeble in the U.S.A. E. coli JM109 Electro-Cells (Cat. #9022) * Competent Cells and Electro-Cells are not availeble in the U.S.A. IPTG (Cat. #9030) X-Gal (Cat. #9031) SUPREC -01 (Cat. #9040) TaKaRa Taq (Cat. #R001A/B/C) TaKaRa Ex Taq (Cat. #RR001A/B/C) TaKaRa LA Taq (Cat. #RR002A) Pyrobest DNA Polymerase (Cat. #R005A/B) * not available in the U.S.A. puc118 Hinc II/BAP (Cat. #3322) Reagent Set for Mighty Cloning Kit (Blunt End) (Cat. #6027) 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 or contact from our website at Phone: Fax: