Zback Faster Ligation Kit

Transcription

1 Zback Faster Ligation Kit For the highest efficiency cloning of PCR products either blunt or sticky-end Kit Contents Contents TGVTB04 TGVTB04-2 pzback/blunt vector 10 µl 20 µl T4 DNA Ligase 5 µl 10 µl 2 Reaction Buffer 50 µl 100 µl DNA Blunting Enzyme 5 µl 10 µl pzback//blunt Forward 100 µl 200 µl pzback//blunt Reverse 100 µl 200 µl Control Insert DNA 10 µl 10 µl ddh 2 O 1 ml 1 ml Storage All components of the ZBack Faster Ligation Kit should be stored at -20 C. Repeated freeze-thaw cycles should be avoided.

2 Introduction TOOLS ZBack Faster Ligation Kit is an advanced positive selection system for the highest efficiency cloning of PCR products generated with Pfu DNA polymerase, Taq DNA polymerase, or other thermo-stable DNA polymerases. Additionally, any other DNA fragment, either blunt or sticky-end, can be successfully cloned using the kit. Cloning is fast and efficient; ligation takes only 5 minutes and yields more than 99% positive clones. The kit features the novel positive selection cloning vector pzback/blunt. This vector contains a lethal gene which is disrupted by ligation of a DNA insert into the cloning site. As a result, only cells with recombinant plasmids are able to propagate, eliminating the need for expensive blue/white screening. The vector contains an expanded multiple cloning site, as well as a T7 promoter for in vitro transcription. Sequencing primers are included for convenient sequencing of the insert. Cloning Principle 1. pzback/blunt is a linearized cloning vector, which accepts inserts from 6 bp to 10 kb. The 5'-ends of the vector cloning site contain phosphoryl groups, therefore, phosphorylation of the PCR primers is not required. 2. Blunt-end PCR products generated by proofreading DNA polymerases can be directly ligated in just 5 min with the pzback/blunt vector. PCR products with 3 -da overhangs generated using Taq DNA polymerase or other non-proofreading thermostable DNA polymerases are blunted in 5 min with a proprietary thermostable DNA blunting enzyme (included in the kit) prior to ligation. All common laboratory E.coli strains can be directly transformed with the ligation product. 3. Re-circularized pzback/blunt vector expresses a lethal restriction enzyme after transformation and is not propagated. As a result, only recombinant clones containing the insert appear on culture plates. Therefore, blue/white screening is not required. 4. The kit performs well over a wide range of insert/vector molar ratios (0.5:1 to 15:1). For insert size less than 1 kb, the insert/vector ratio is 3:1. For insert size more than 1 kb, the insert/vector ratio is 7:1. Cloning Protocols Blunt-End Cloning Protocol Use for cloning blunt-end PCR products generated by proofreading DNA polymerases, such as Pfu DNA polymerase. If the end structure of the PCR products is not specified by the supplier of the DNA polymerase, follow the Sticky-End Cloning Protocol. This protocol can also be used for cloning blunt-end DNA fragments generated by restriction digestion. Gel-purify the DNA fragment prior to ligation and use in a 3:1 molar ratio with pzback/blunt vector. 1

3 All procedure must be done in asepsis environment. 1. Set up the ligation reaction: PCR product X μl 2 Reaction Buffer 5 μl pzback/blunt vector 1 μl (25 ng/µl) T4 DNA Ligase (5 u/µl) ddh 2 O Up to 10 μl 2. Incubate the ligation mixture at room temperature (22 C) for 5 min. Note: Incubation time can be extended up to 30 min if the insert fragments are larger than 3 kb. 3. Use the ligation mixture directly for bacterial transformation (see p.6). Sticky-End Cloning Protocol Use for cloning PCR products with 3'-dA overhangs generated by Taq DNA polymerase or enzyme mixtures containing Taq DNA polymerase. If the end structure of the PCR products is not specified by the supplier of the DNA polymerase, follow the Sticky-End Cloning Protocol. This protocol can also be used for cloning DNA fragments with 5 - or 3 -overhangs generated by restriction digestion. Gel-purify the DNA fragment prior to ligation and use in a 3:1 molar ratio with pzback/blunt. The DNA blunting enzyme is a proprietary thermostable DNA polymerase with proofreading activity. It will remove 3'-overhangs and fill-in 5'-overhangs. Nucleotides for the blunting reaction are supplied in the reaction buffer. All procedure must be done in asepsis environment. 1. Set up the ligation reaction: PCR product X μl 2 Reaction Buffer 5 μl DNA Blunting Enzyme (5 u/µl) ddh 2 O Up to 8.5 μl 2

4 2. Incubate the mixture at 70 C for 5 min. Chill briefly on ice. 3. Set up the ligation reaction. Add the following to the blunting reaction mixture: pzback/blunt vector (25 ng/µl) T4 DNA Ligase (5 u/µl) 1 μl 4. Incubate the ligation mixture at room temperature (22 C) for 5 min. Note: Incubation time can be extended up to 30 min if the insert fragments are larger than 3 kb. 5. Use the ligation mixture directly for bacterial transformation (see p.8). Control Experiment Follow the Sticky-End Protocol 1. Set up the ligation reaction: 2 Reaction Buffer 5 μl Control PCR product (700 bp, 50 ng/ μl) DNA Blunting Enzyme ddh 2 O Up to 8.5 μl 2. Incubate the mixture at 70 C for 5 min. Chill briefly on ice. 3. Set up the ligation reaction. Add the following to the blunting reaction mixture: pzback/blunt vector (25 ng/µl) T4 DNA Ligase (5 u/µl) 1 μl 4. Incubate the ligation mixture at room temperature (22 C) for 5 min. 5. Use the ligation mixture directly for bacterial transformation (see p.4). 3

5 Transformation of competent E.coli cells 1. Prepare LB-ampicillin agar plates (with a finial ampicillin concentration of 100 µg/ml). Pre-warm the plates at 37 C for at least 20 min. 2. Transformation step i. Remove tube(s) of TOP10 competent cells from storage and place in an ice bath until just thawed. Carefully add part of ligation-reaction mixture to µl TOP10 competent cells. The adding volume of ligation-reaction mixture should be less than one tenth of competent cell volume. Gently flick the tubes to mix and place them on ice for 30 min. At the same time, use control plasmid puc19 to transform competent cell to detect transformation efficiency. Add 1 μl puc19 to another tube with proper competent cell, and then, other steps go along with the step of transformation of ligation product during the same period. ii. Heat-shock the cells for 90 s in a water bath at exactly 42 C (do not shake). Immediately return the tubes to ice for 2-3 min (do not shake). iii. Add μl room temperature SOC or LB culture medium per tube (not containing antibiotic), and then incubate for 45 min at 37 C with shaking (~150 rpm). iv. Mix bacterium in the tube completely. Then plate 100 μl transformation cultures onto each LB agar plate containing antibiotic to ensure good separation of colonies for subsequent single-colony isolation. Smear bacterium completely with asepsis elbow glass stick. After the surface of plate is dry, then put the plate at 37 for hours. 3. Detection a. General detection: pipet the transformation mixture into1-5 ml liquid LB culture medium (containing μg/ml ampicillin), and culture at 37 overnight with shaking. Save bacterium strain and extract plasmid. To detect whether the fragment has inserted rightly using PCR or enzyme cutting. For PCR detection of Control Insert DNA, the follow program can be used: 94 3 min sec sec 30 cycles 65 1 min 65 5 min b. Quick detection: to detect whether the fragment has inserted rightly using bacterium PCR directly. c. Sequencing: sequence the fragment after general or quick detection. Forward Sequencing Primer, 23-mer Reverse Sequencing Primer, 24-mer 5 -CGACTCACTATAGGGAGAGCGGC-3 5 -AAGAACATCGATTTTCCATGGCAG-3 The product is for research only, not for diagnostic and clinical use. 4

6 pzback/blunt Vector Map pzback/blunt 2974 bp pzback/blunt Vector Cloning site 5