Rapid DNA Ligation & Transformation Kit

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1 1 Rapid DNA Ligation & Transformation Kit (#K1432 for 30 reactions) INTRODUCTION The Rapid DNA Ligation & Transformation Kit enables ligation of any type of DNA in 5 minutes at ambient temperature followed by a fast transformation procedure. The Rapid Ligation Buffer is optimized for quick and effi cient ligation of cohesive-ended or blunt-ended DNA fragments [1-3]. The ligation reaction mixture can be used directly to transform competent cells according to the suggested procedure. The TransformAid Bacterial Transformation System is included in this kit for the preparation of competent cells that have consistently high transformation effi ciency. In order to avoid self circularization and/or formation of tandem oligomers of insert and linearized vector, the optimum insert to vector molar ratio must be determined. Use the following guidelines to estimate your molar ratios: pmol ends = pmol DNA x (number of cuts x 2 + 2), 1µg of 1000 bp DNA = 3.04pmol ends, 1µg of linear puc18/19 DNA = 1.14pmol ends, 1µg of linear pbr322 DNA = 0.7pmol ends, 1µg of linear SV40 DNA = 0.58pmol ends, 1µg of linear ΦX174 DNA = 0.56pmol ends, 1µg of linear M13mp18/19 DNA = 0.42pmol ends, 1µg of λ phage DNA = 0.06pmol ends. Ligation reaction mixture should contain >1-3 fold molar excess of foreign DNA termini to vector DNA. 2 Recircularization of plasmid DNA can be minimized by the removal of the 5 -phosphates from both termini of cleaved vector DNA with Calf Intestinal Alkaline Phosphatase [5] or Shrimp Alkaline Phosphatase. Another factor which may affect ligation and transformation is purity of DNA. If you chose to purify by agarose gel electrophoresis, ensure that high agarose quality is used since some preparations may contain DNA ligase inhibitors that are diffi cult to remove. For recovery of DNA fragments from agarose gels we recommend DNA Extraction Kit (#K0513) or Agarase (#EO0461). COMPONENTS OF THE KIT 1. T4 DNA Ligase 5u*/µl 30µl of enzyme in storage buffer containing 20mM Tris-HCl (ph 7.5), 1mM DTT, 50mM KCl, 0.1mM EDTA, 50% glycerol. 2. 5X Rapid Ligation Buffer**: 0.6ml of ready-to-use solution. 3. Water, nuclease-free: 1.5ml of 0.22µm membrane-fi ltered molecular biology grade water. 4. TransformAid Bacterial Transformation System***: a) 2x35ml of C-Medium. b) 5ml (4x1.25ml) T-Solution (A). c) 5ml (4x1.25ml) T-Solution (B). * One unit of enzyme catalyzes the conversion of 1nanomole of [ 32 PP i ] into Norit -adsorbable form in 20min at 37 C (Weiss unit). ** The Buffer can be thawed and frozen repeatedly. It is absolutely necessary to thoroughly mix the buffer prior to use. *** Does not contain glucose.

2 3 PROTOCOL 1. Ligation 1.1. Insertion of Foreign DNA into Plasmid Vector! To a microcentrifuge tube, add correct ratio (see Note 1) of digested, and if preferred dephosphorylated and purifi ed, plasmid (50-100ng) and foreign DNA to be inserted in 5-10µl of water or TE buffer, ph 7.8. Digested plasmid or DNA fragment solution can be directly used for ligation if digestion was performed in Fermentas Five Buffer System. " Add the following components to the same tube: 5X Rapid Ligation Buffer 4µl Water, nuclease-free, up to 19µl T4 DNA Ligase (5u) 1µl Vortex the tube and spin down in a microcentrifuge for 3-5sec. # Incubate the mixture for 5 min at 22 C. To achieve a maximum yield of recombinants, incubation may be prolonged for up to 1 hour. $ Use 2-5µl of the ligation mixture for transformation (see Note 2). The reaction mixture can be stored at 0-4 C until used for transformation. Typical Experiment Blunt ended DNA fragment (approx bp length) was extracted from agarose gel using DNA Extraction Kit (#K0513) or Agarase (#EO0461). The mixture containing the DNA fragments 130ng (0.17pmol ends) and dephosphorylated puc19 DNA/SmaI digest 50ng (0.057pmol ends) was prepared and ligation reaction was performed as described above. The E.coli XL1-Blue competent cells, that were prepared using TransformAid Bacterial Transformation System resulted in 10 7 colonies/µg of supercoiled puc19 DNA. Yield of white colonies after transformation into competent cells is 10 5 colonies/µg DNA. 4 Analysis of ligation products by agarose gel electrophoresis Ligation mix (10µl of the ligation product) can be analyzed by agarose gel electrophoresis by the addition of 2µl of 6X loading solution (0.09% bromophenol blue, 0.09% xylene cyanol FF, 60% glycerol, 60mM EDTA, 1% SDS). Before loading on to a gel heat the sample at 65 C for 10 min. Note. 1) Ligation reaction mixture should contain >1-3 fold molar excess of foreign DNA termini to vector DNA. The maximum amount of vector DNA is 200ng. Ligation effi ciency may decrease as insert size increases. 2) The ligation mixture can be used directly to transforming competent cells. A maximum 5µl of the ligation reaction can be used for transformation Recircularization of Linear DNA! Prepare 10-35µl solution of linear DNA (25-50ng) in water or TE buffer, ph 7.8 in a microcentrifuge tube. " Add the following components to the same tube: 5X Rapid Ligation Buffer 10µl Water, nuclease-free, up to 49µl T4 DNA Ligase (5u) 1µl # Vortex and spin down in a microcentrifuge for 3-5sec. Incubate the tube at 22 C for 5 min. $ Use 2-5µl of the ligation mixture for transformation (see Note 1). The reaction mixture can be stored at 0-4 C until used for transformation.

3 5 Typical Experiment All the components described above were added to 30µl of aqueous solution containing 50ng SmaI digested puc19 DNA. The ligation reaction was performed as described above. 1µl (1ng DNA) of the mixture was added to 50µl of E.coli XL1-Blue competent cells, that were prepared using TransformAid Bacterial Transformation System. Approximately 3.1 x 10 6 colonies/µg DNA were obtained. Note. 1) Ligation reaction mixture should contain >1-3 fold molar excess of foreign DNA termini to vector DNA. The maximum amount of vector DNA is 200ng. Ligation effi ciency may decrease as insert size increases. 2) The ligation mixture can be used directly to transforming competent cells. A maximum 5µl of the ligation reaction mixture can be used for transformation. 2. Transformation using the TransformAid Bacterial Transformation System 2.1. Preparation of Bacteria from Overnight Bacterial Culture! Inoculate 2ml of TransformAid C-Medium with bacteria from a frozen stock or a colony. Incubate the culture at 37 C overnight. " Pre-warm culture tubes containing the required amount of TransformAid C-Medium (1.5ml for each of 2 transformations) at 37 C. # Add 1/10 volume of the overnight culture to the pre-warmed C-Medium (0.15ml overnight culture for each 1.5ml C-Medium). Incubate the tubes in a shaker at 37 C for 20min. Note. A maximum of 26 transformations can be performed from 2ml of overnight culture. The culture can be kept at 4 C for at least a week and used for preparation of fresh cultures Preparation of Bacteria from Bacterial Culture Plate! Inoculate a LB plate with bacteria using streak plate method, and incubate the plate at 37 C overnight. " Pre-warm culture tubes containing the required amount of TransformAid C-Medium (1.5ml for each 2 transformations) at 37 C. # Move a small portion of bacterial culture (4 x 4mm size for each 1.5ml of C-Medium) from the overnight LB plate using inoculating loop in to the pre-warmed C-Medium. Suspend the culture by gently mixing and incubate the tubes in a shaker at 37 C for 2 hours. Note. The colonies on LB plates can be stored at 4 C and used for inoculating fresh cultures within 10 days Transformation Procedure! Pre-warm LB-Ampicillin agar plates (see Note 1) in a 37 C incubator for at least 20min. " Prepare TransformAid T-Solution by mixing equal volumes of T-Solution (A) and T-Solution (B) (500µl of solution for each 2 transformations). Keep TransformAid T-Solution on ice (see Note 2). # Dispense 1.5ml of fresh culture into a microcentrifuge tube and spin at maximum speed for 1min at room temperature (RT) or at 4 C (see Note 3). $ Discard the supernatant and resuspend the pelleted cells in 300µl of TransformAid T-Solution. Incubate the tubes on ice for 5min. % Spin down the cells again for 1min at RT or at 4 C and then remove the supernatant. & Resuspend the cells in 120µl of TransformAid T-Solution and incubate on ice for 5min.

4 7 ' Prepare DNA for transformation (by dispensing 1µl of supercoiled DNA (10-100pg) or up to 5µl of ligation mixture (10-100ng of vector DNA) into new microcentrifuge tubes and sit them on ice for 2min (see Note 4). ) Add 50µl of the resuspended cells to each tube containing DNA and incubate them on ice for 5min. * Plate the cells on pre-warmed LB-Ampicillin agar plates. Incubate the plates overnight at 37 C. Note. 1) DNA containing other antibiotic resistance genes can be used in the TransformAid system. The transformation effi ciency should fi rst be tested with supercoiled DNA containing these antibiotic genes. In our hands, DNA containing tetracycline and chloramphenicol resistance genes have similar transformation effi ciency as DNA containing the ampicillin resistance gene (~10 6 to 10 7 ). 2) After thawing T-Solution (A) and T-Solution (B), mix contents thoroughly prior to combining equal volumes of T-Solution (A) and T-Solution (B). 3) The centrifugation can be carried out at room temperature (RT), but the cells should be kept on ice at all other times as indicated. Do not leave the cells in the centrifuge at RT for more than 5min as this will signifi cantly decrease the transformation effi ciency. 4) The quantity of DNA used in transformation will infl uence the transformation effi ciency. The transformation effi ciency usually decreases with an excess of DNA. In the TransformAid system, approximately 100 to 2000 colonies will be obtained using 100pg of supercoiled DNA for most bacterial strains (DH5α, JM107, JM109, SURE, TOPP2, W3110, NM527, AD494 and CJ236). For the best effi ciency of competent cells only freshly plated bacterial culture 8 should be used for overnight inoculation, especially when preparing DH5α competent cells. To transform cells with ligation reaction mixture, a reaction volume not larger than 5µl should be used Scale-up When preparing a large volume of competent cells for transformation, the transformation procedure can be scaled up proportionately. The cells can be pelleted in large centrifuge tubes at 5,000-10,000xg for 5 minutes in steps 3 and 5 of transformation procedure Preparation of LB-Ampicillin Plates! Prepare LB Medium (per liter): Peptone 10g Yeast extract 5g NaCl 10g " Dissolve solids in 800ml of water, adjust ph to 7.5 with NaOH. # Adjust water to a fi nal volume of 1000ml. $ Autoclave the medium. Prepare LB-Ampicillin plates: % Adjust 15g agar to 1 liter of LB medium. & Microwave the medium until the agar dissolves. ' Allow the solution to cool down to 55 C, then add ampicillin to a fi nal concentration of 50µg/ml. (For convenience, prepare a stock solution of ampicillin at 25mg/ml. Use 2ml of the stock solution per liter of LB-agar medium. Store the stock solution at -20 C.) ) Mix gently, and pour 30-35ml of LB -agar medium directly onto each plate.

5 9 IPTG (isopropyl-β-d-thiogalactopyranoside) (#R0391, #R0392, #R0393) stock solution (0.1M): 1.2g IPTG, add water to 50ml fi nal volume. Filter-sterilize and store at 4 C. Use 40µl per plate. X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) (#R0401, #R0402, #R0404) stock solution: 20mg/ml in N,N-dimethylformamide. Use 40µl per plate. For other antibiotics, use the following concentrations: Tetracycline, 12µg/ml; Chloramphenicol, 20µg/ml. Note. The concentration of antibiotic in the agar plates will affect the number of colonies obtained. The antibiotic resistance of transformants is dependent on the copy number of the plasmid used. When a low copy number plasmid is used, the concentration of the antibiotic can be decreased in order to obtain a greater number of transformants. QUALITY CONTROL Each lot of the kit is pre-tested, using 50ng of puc19 DNA, digested with SmaI, and dephosphorylated with alkaline phosphatase, in a ligation reaction with 130ng of blunt-ended 2300bp insert DNA according to the standard protocol. Yield of white colonies after transformation into competent E.coli XL1-Blue cells >1x10 5 per µg DNA. Quality authorized by: Ramute Pineliene 10 Problem Few or no transformants High background of nonrecombinants TROUBLESHOOTING Possible solution a. Experimental DNA contains an inhibitor of ligation. Ensure DNA is free of contaminants (e.g. excess salts, EDTA, proteins, phenol, etc.) that may inhibit ligation. Gel purity and/or extract the vector and insert prior to ligation. b. Vector and/or insert have been damaged. Minimize UV exposure. c. Incompatible ends. Recheck cloning strategy. d. Incorrect vector:insert ratio. Use correct ratio. e. Cloned sequence is not tolerated in E.coli. If possible, check the target sequence for strong E.coli promoters or other potencially toxic elements, as well as inverted repeats. In case the product of a cloned gene is detrimental to a host, use promoters with a very low expression background. f. Use only freshly plated bacteria for inoculation to obtain cells of highest transformation effi ciency. a. Supply LB medium with fresh antibiotic. b. Dephosphorylation of the vector helps increase ligation effi ciency by eliminating recircularization of the vector. For ligation into dephosphorylated vectors, the insert must have 5 -phosphate groups.

6 11 References 1. Pfeiffer, B.H. and Zimmerman, S.B., Nucl. Acids Res., 11, 7853, Hayashi, O., et al., Nucl. Acids Res. vol. 13, No 22, 7979, Bercovich, J.A., et al., BioTechniques, vol. 12, No 2, 190, Michelsen, B.K., Anal. Biochem., 225,172, Sambrook, J., and Russel D.W., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, N.Y., For Note: Trademarks Norit is a registered trademark of American Norit Company. TransformAid is a trademark of Fermentas. DH5α is a registered trademark of Life Technologies Inc. SURE and TOPP2 are registered trademarks of Stratagene Cloning System 3 Revised

7 13 Related Products Calf Intestine Alkaline Phosphatase #EF0341 #EF0342 Shrimp Alkaline Phosphatase #EF0511 Agarase #EO0461 DNA Extraction Kit #K0513 Top Vision LM GQ Agarose #R0801 Top Vision LE GQ Agarose #R0491 IPTG #R0391 #R0392 #R0393 X-Gal #R0401 #R0402 #R0404 PRODUCT USE LIMITATION. This product is developed, designed and sold exclusively for research purposes and in vitro use only. The product was not tested for use in diagnostics or for drug development, nor is it suitable for administration to humans or animals. Please refer to for Material Safety Data Sheet of the product.