Table of contents. I. Flowchart of blunt end cloning of PCR products...2. II. Description...3. III. Kit Components...3

Transcription

1 Table of contents I. Flowchart of blunt end cloning of PCR products...2 II. Description...3 III. Kit Components...3 IV. Reagents and Instruments Required...3 V. Storage...3 VI. About puc118 Hinc II/BAP...4 VII. Protocols...5 VII-1. Procedure...5 VII-2. Note...7 VIII. Appplication Example...8 IX. Comparison among PCR enzymes...8 X. Related products...9 1

2 I. Flowchart of blunt end cloning of PCR products : PCR amplification of target gene PCR products with blunt end e.g. obtained with PrimerSTAR HS DNA Polymerase e.t.c. PCR products with the end added with da e.g. obtained with TaKaRa Taq TaKaRa Ex Taq TaKaRa Ex Taq Hot Start Version TaKaRa LA Taq e.t.c. Verify amplified products through electrophoresis Single band When primer dimer generates When extra band appears Directly apply PCR products to cloning Simply purify PCR products Recover target bands from agarose gel after gel electrophoresis (Related purification products) SUPREC -01 EASYTRAP Ver.2 TaKaRa RECOCHIP * Cloning with Mighty Cloning Kit <Blunt End> * : Other blunt-end vectors than supplied puc118-hinc II/BAP are available. Transformation into E. coli Selection of target clones 2

3 II. Description : Takara's Reagent Set for Mighty cloning Kit (Blunt End) is designed to allow that PCR products are cloned into blunt-end vectors in a simple method and quickly. As PCR products are blunt-ended in parallel with phosphorized at 5'-end, not only from bluntend PCR products, but also from products added with da at termini, 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 <Mighty mix>, which achieves the series of operation efficiently and in a short time. III. Kit Components (20 reactions) : Blunting Kination Buffer μl 2. Blunting Kination Enzyme Mix 20 μl 3. Ligation Mighty Mix * μl 4. puc118-hinc II/BAP 50 ng/μl 20 μl 5. Control Insert * ng/μl 10 μl 6. ddh2o 500 μl * 1 : Ligation Mighty Mix is the same reagent that is included in DNA Ligation <Mighty Mix> (Cat. #6023). * 2 : 500 bp PCR fragment amplified with TaKaRa Ex Taq by using λ-dna as a template. IV. Reagents and Instruments Required but Not Supplied in the Kit : Competent cell or Electro cell SOC culture medium Ampicillin/X-Gal/LB plate added with IPTG V. Storage : -20 3

4 VI. puc118 Hinc II/BAP : puc118 DNA (BAP-treated) is a useful blunt-end clonig vector, prepared by cleavage with Hinc II, then dephosphorylated by alkaline phosphatase purified from E. coli (BAP). The sequence of DNA cloned into this vector can be verified with M13 primer M4 and RV. Vector map of puc118 Cloning site of puc lacz 287 G A A T T C G A G C T C G G T A C C C G G G G A T C C T C T A G A G T C G A C C T G C A G G C A T G C A A G C T T G G C 3 EcoR I Kpn I BamH I Pst I Hind III Sac I Sma I Xba I Sph I Xma I Sal I Sse8387 I Acc I Hinc II Blunt Cloning Site 4

5 VII. Protocol : VII-1. Procedure A. When PCR reactant is directly used : Blunting Kination reaction 1. Prepare the following reaction mixture in a micro tube. PCR reactant * < 2 μl 10 Blunting Kination Buffer 2 μl Blunting Kination Enzyme Mix 1 μl ddh2o X μl Total 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 at12,000 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 1 μl of puc118 Hinc II/BAP (50 ng/μl), and mix. 14. Add the equivalent volume of Ligation Mighty Mix and mix gently. 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. 17. Apply to the LB plate including Ampicillin, X-Gal and IPTG. * 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 2 μl. When large volume is used, the reaction efficiency may lower. Please exchange the buffer by ethanol precipitation when more PCR products are applied. * When a plasmid having the same selection marker that a cloning vector has is used as a template of PCR, it is necessary to prevent generating colonies having the template plasmid itself. Therefore, purification of target band from agarose gel is recommended after gel electrophoresis. 5

6 B. When purified DNA fragment is used : Blunting Kination reaction 1. Prepare the following reaction mixture in a micro tube. DNA Fragment pmol 10 Blunting Kination Buffer 2 μl Blunting Kination Enzyme Mix 1 μl ddh2o X μl Total 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 at12,000 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 1 μl of puc118 Hinc II/BAP (50 ng/μl), and mix. 14. Add the equivalent volume of Ligation Mighty Mix and mix gently. 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. 17. Apply to the LB plate including Ampicillin, X-Gal and IPTG. C. Control experiment : 1. Prepare the following reaction mixture. Control Insert 10 Blunting Kination Buffer Blunting Kination Enzyme Mix ddh2o Total 2 μl 2 μl 1 μl 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

7 6. Centrifuge at12,000 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. Put 5 μl of DNA solution obtained at step 5 into a new micro tube. 13. Add 1 μl of puc118 Hinc II/ BAP (50 ng/μl), and mix. 14. Add 6 μl of Ligation Mighty Mix and mix gently. 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. 17. Apply to the LB plate including Ampicillin, X-Gal and IPTG. When the E. coli JM109 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. VII-2. Note 1. Thaw Ligation Mighty Mix on ice or in ice water. Mix gently before use. Ligation Mighty Mix would not be inactivated by freeze-thaw cycles. 2. When ligation efficiency is low, extend the ligation reaction time to overnight. When the above remedy 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. 7

8 VIII. Application Example : Cloning of various size fragment : (A) When PCR products are directly used Various size fragments (500 bp, 1 kb, 2 kb, 4 kb, and 6 kb) were amplified with PrimeSTAR HS DNA Polymerase by using λ-dna as template. Each 2 μl of each PCR reactant were blunted, phosphorylated, cloned into puc118 Hinc II/BAP, and then transformed in E. coli JM109 Competent cells for color selection. The existence of insert were verified through PCR by using primers: M13 Primer M4 and RV. Insertion DNA Chain length (bp) White colony/blue colony (/50 ng puc118 DNA) Insert DNA/White colony / /10 1, / /10 2, / /10 4, / /10 6, / /10 * Transformation efficiency of the used E. coli JM109 Competent cells : colonies/μg puc118 DNA (B) When using DNA fragments recovered from agarose gel The 2 DNA fragments were amplified as follows; 500 bp obtained with TaKaRa Ex Taq by using λ-dna as template, and 2 kb amplified with PrimeSTAR HS DNA Polymerase by using human genomic DNA as template. They were applied to agarose gel electrophoresis after PCR, and were recovered with EASYTRAP Ver.2. Each of the DNA were blunted and phosphorylated with this kit, in 2 pmol (approx. 660 ng) about the 500 bp fragment, and in 0.2 pmol (approx. 250 ng) about the 2 kb gragment. Then they were cloned into puc118 Hinc II/BAP and were transformed in E. coli JM109 competent cells for color selection. The existence of insert were verified through PCR by using primers: M13 Primer M4 and RV. Insertion DNA Chain length (bp) White colony/blue colony (/50 ng puc118 DNA) Insert DNA/White colony / /10 2, / /10 * Transformation efficiency of the used E. coli JM109 Competent cells : colonies/μg puc118 DNA 8

9 IX. Comparison among PCR enzymes : The 1 kb of PCR products were prepared with TaKaRa Taq, TaKaRa Ex Taq, TaKaRa LA Taq, PrimeSTAR HS DNA Polymerase and TaKaRa EX Taq Hot Star Version using λ-dna as a template. Following the protocol of this kit, they were cloned into Hinc II/ BAP site of puc118. Then the transformation was done using E. coli JM109 and then color selection was performed. The existence of insert were verified through PCR by using primers: M13 Primer M4 and RV. DNA Polymerase used for PCR White colony/blue colony (/50 ng puc118 DNA) Insert DNA/White colony PrimeSTAR HS DNA Polymerase / /10 TaKaRa Taq / /10 TaKaRa Ex Taq / /10 TAKaRa LA Taq / /10 TaKaRa Ex Taq Hot Start Version / /10 * Transformation efficiency of the used E. coli JM109 Competent cells : colonies/μg puc118 DNA X. 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. TaKaRa RECOCHIP (Cat. #9039) SUPREC 01 (Cat. #9040) EASYTRAP Version 2 (Cat. #9410) TaKaRa Taq (Cat. #R001A/B/C) TaKaRa Ex Taq (Cat. #RR001A/B/C) TaKaRa LA Taq (Cat. #RR002A) TaKaRa Ex Taq Hot Start Version (Cat. #RR006A/B) PrimeSTAR HS DNA Polymerase (Cat. #R010A/B) PrimeSTAR HS DNA Polymerase with GC Buffer (Cat. #R044A/B) puc118 Hinc II/BAP (Cat. #3322) One Shot Insert Check PCR Mix (Cat. #RR010A) PerfectShot Insert Check PCR Mix (Cat. #RR020A) 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: