User Manual
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1. Molecular Cloning Laboratories lt MCLAB Version 3 2 User Manual Product name Choo Choo Cloning Kits Cat CCK 10 CCK 20 CCK 096 CCK 384 Description Choo Choo Cloning is a highly efficient directional PCR cloning kit designed for rapid cloning of one or multiple PCR fragments without the need for ligase or restriction enzyme It enables to clone any PCR fragment into any linearized vector at any location By simply incubating on ice the ends of a PCR generated DNA fragment can be precisely fused into a DNA vector with 6 bp or more overlapping homologous sequence The system is very robust up to 8 PCR generated DNA fragments can be assembled into one piece up to 10 kb and cloned into a vector of choice in one step The system is highly efficient with 98 100 positive colonies The function of Choo Choo Cloning depends on MCLAB s proprietary enzyme systems There is no need for restriction enzyme digestion ligation and blunt end polishing Any extra unwanted bases can be eliminated from the final construct The linearized vector can be generated by PCR or restriction enzyme digestion The PCR fragments can be generated by Tag DNA polymerase or other high fidelity DNA polymerase The addition of an A by Taq DNA polymerase is not required or has no effect on cloning efficiency If the PCR product is amplified from a plasmid template a gel purification step is needed to reduce the background In addition to PCR clon2. O for a total volume of 100 ml Filter sterilize this solution 7 Let the autoclaved solutions cool to about 55 C then add 10 ml of filter sterilized 2 M glucose solution and 10 ml of 1 M MgCl Store at room temperature or 4 C PCR and Experimental Preparation PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING A PCR Primer Design Primer design and quality are critical for the success of Choo Choo Cloning reaction Two or more fragments e g vector and insert or multiple inserts can be joined together as long as they share 6 20 bases of homology at each end The best result can be obtained from 12 15 bp overlapping range Figure 2a outlines the guidelines for universal primer design for vector using single restriction site Figure 2b outlines the guidelines for universal primer design for vector using double restriction sites Every Choo Choo Cloning primers must serve two purposes it should contain a homologous sequence and be gene specific The 6 20 base pairs towards the 5 end of the primer must match the 6 20 base pairs at the linear end of the DNA fragment to which it will be joined The 3 end of the primer is the gene specific portion of the primer The 3 end of the primer must have a melting temperature Tm suitable for PCR Please note that the Tm should be calculated based upon the 3 gene specific end of the primer and not 1 650 872 0245 www mclab com 3 the entire primer If the calculated Tm is too
3. ing the Choo Choo Cloning Kit is versatile and can be used for other applications such as adaptor linker and tag addition before and after the inserts and gene synthesis List of Components Cat Box1 Box 2 Choo Choo Cloning 10x Choo Choo Cloning Choo Choo Blue chemical Enzyme Mix Reaction Buffer Competent cell CCK 10 10 reactions 20 ul 20 ul 10 tubes x 50 ul CCK 20 20 reactions 40 ul 40 ul 20 tubes x 50 pl CCK 100 100 reactions 200 ul 200 yl 100 tubes x 50 ul Store Box 1 at 20 C Store Box 2 at 80 C Figure The Choo Choo Cloning Method During the 45 min incubation the Choo Choo Cloning enzyme mix containing MCLAB s proprietary enzymes and recombinant proteins creates single stranded regions at each end of the vector and PCR fragments which are then linked together due to the 6 20 bp overlapping homologous sequences The resulting construct can be directly transformed into E co i competent cells 1 650 872 0245 www mclab com 1 Choo Choo Cloning Kits 1 Generate a linear vector 2 PCR amplify DNA inserts up to 8 with overlapping ends 3 Mix vector PCR fragments and Choo Choo Cloning enzyme mix in a tube and incubate on ice for 45 min z 8 Incubate on ice 45 min 4 Transformation Figure 1 Experimental workflow of mutiple PCR fragments insertion into a vector using the Choo Choo Cloning Kit lt MCLAB Additiona
4. l Materials Required The following materials are required but not supplied LB Luria Bertani medium pH 7 0 e 1 0 Bacto tryptone 10g e 0 5 Yeast extract 5g e 1 0 NaCl 10g 1 For 1 liter dissolve ingredients in 950 ml of deionized H O Adjust the pH to 7 0 with 5M NaOH and bring the volume up to 1 L Autoclave on liquid cycle for 20 min at 15 Ib in2 Store at room temperature or at 4 C LB antibiotic plates 2 Prepare LB media as described above then add 15 g L of agar in to LB media Autoclave on liquid cycle for 20 min at 15 Ib in2 Let cool to 55 C add antibiotic e g 100 ug ml of ampicillin and pour into 10 cm plates After the plates hardened invert and store at 4 C SOC medium 2 Tryptone 0 5 Yeast Extract 10 mM NaCl 2 5 mM KCl e 10 mM MgCl 6H O e 20mM glucosa 1 For 1 liter dissolve 20 g of tryptone 5 g of yeast extract and 0 5 g of NaCl in 950 ml of deionized H O 2 Prepare a 250 mM KCI solution by dissolving 1 86 g of KCI in deionized H O for a total volume of 100 ml Add 10 ml of this stock KCI solution to the solution prepared in Step 1 3 Adjust pH to 7 0 with 5 M NaOH and then bring the volume to 980 ml with deionized H O 4 Prepare a 1 M solution of MgCl by dissolving 20 33 g of MgCl e6H O in deionized H O for a total volume of 100 ml 5 Autoclave both solutions on liquid cycle at 15 Ib in2 for 20 min 6 Meanwhile make a 2 M solution of glucose by dissolving 36 g of glucose in deionized H
5. low increase the length of the gene specific portion of the primer until a Tm of between 58 65 C is reached The Tm difference between the forward and reverse primers should be lt 4 C otherwise a good amplification will not be achieved Desalted oligos in PCR reactions are recommended to use Figure 2 Guidelines for universal primer design 4 Forward Primer With 6 20 sequence homology to vector Restriction Gene specific Vector sequence 1 enzyme sequence With 6 20 sequence homology to vector Gene specific Restriction sequence enzyme Vector sequence Reverse Primer 2a For vector using single restriction site Forward Primer With 6 20 sequence homology to vector Gene specific Restric ion sequence Restriction Vector sequence enzyme Reverse Primer Vector 2b For vector using double restriction sites lt gt MCLAB B Preparation of linearized Vector by Restriction Digestion To carry out a successful Choo Choo Cloning experiment a pure linearized vector must be generated first with very low background of uncut vector present Restriction enzymes may introduce certain amount of background due to difference in cutting efficiencies In general two enzymes cut better than any single enzyme We recommend to select two enzymes to linearize the vector Efficiency of the digestion is always better if the restriction enzyme sites are as far apart as possible I
6. merase When PCR cycling is complete analyze the PCR product by electrophoresis on an agarose EtBr gel to confirm that a correct DNA fragment is obtained and to estimate the concentration of the PCR product Quantify the amount of DNA by measuring against a known standard or molecular weight marker ladder run on the same gel Choo Choo Cloning Reaction and Transformation Procedure 1 Set up the following 15 ul Choo Choo Cloning reaction Reagent Volume Amount 10X Choo Choo Reaction Buffer 1 5ul Choo Choo Cloning Kit Enzyme Mix 2ul Linearized Vector 40 100 ng Insert 5ul Water Up to 15ul The linearized vector can be obtained from PCR or by restriction enzyme digestion The optimized ratio of insert to vector is 2 5 1 1 650 872 0245 www mclab com w 3 CN OY Ui 6 Incubate the reaction on ice for 45 min Apply the whole reaction content to 50 pl Choo Choo Golden Chemical Competent E coli cells and incubate on ice for 15 min Heat shock the cells for 1 min at 42 C without shaking then incubate on ice for 2 min Add 200 ul of room temperature S O C media to the cells Cap the tubes and shake at 37 C for 1 hour Spread the whole content from each transformation on pre warmed LB plates with respective antibiotics Incubate plates overnight about 16 h at 37 C Pick 10 defined colonies for analysis lt gt MCLAB
7. n addition increasing the enzyme digestion time and digestion reaction volume will reduce the background Prepare a linearized vector as follows 1 We recommend cutting the vector with two different enzymes to reduce background unless there is only one site available for cloning Vector 2 5 ug 10x Enzyme buffer 5 ul Restriction enzyme 2 5 5 0 Units Deionized water to 50 ul 2 Incubate the restriction digest as directed by restriction enzyme suppliers For many enzymes incubation from 3 hours to overnight can increase linearization and reduce background 3 After digestion purify the linearized vector using an available gel extract kit Control Check the background of the vector by transforming 5 10 ng of the linearized and purified vector into Choo Choo Blue Competent Cells see the following transformation procedure If the background is high continue digesting the vector for a longer period of time after additional amount of restriction enzyme s added Incubate the digestion from 2 hours to overnight then gel purify the remainder of the vector and transform into E co i competent cells C PCR Amplification of Insert It is important to use only 10 50 ng of plasmid DNA as a PCR template However if a pool of cDNA to be amplified the amount of template DNA depends on the relative abundance of the target message in the mRNA population For the best results we recommend using Pfu AFU DNA Polymerase and other high fidelity poly
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