pEcoli Expression Systems User Manual
Contents
1. E o N pEcoli Expression Systems User Manual Clontech United States Canada 800 662 2566 Asia Pacific 1 650 919 7300 Europe 33 0 1 3904 6880 Japan SOTO LORS OMG Cat Nos 631417 amp 631418 Clontech Laboratories Inc PT5018 1 01 251 2 ATakara Bio Company 1290 Terra Bella Ave Mountain View CA 94043 Technical Support US E mail tech clontech com www clontech com pEcoli Expression Systems User Manual Table of Contents I Introduction amp Protocol Overview e xxii iiu Y ENA KNARNNARESERSSWRAAREENNNAEURRWNARNN AA ES ANN EN ARRNR ORAU 3 ll List of Components 9 99WYnubYLL NEL REEADNEEENEEREEAENEREAEENAYEERDEERNREENREERNDEENRERNUEENNDERRRERNRDEREEER 6 lll Additional Materials Required cccccccccceceeeeeeeeeseeeceeeeeeeaeesaneceasaesaeseesaesensenseeesaneaenees 7 M a e 8 V General COSI era ioli Sn ee oes sae ect ar cea cee Y RC ae eee enc bcos ava NR YNAT 9 VI Traditional Cloning Protocol ccccccccceceeeeeeeeeeeceeeeeaeeecaaeesaeeeseeeeseaeesaaseseaaeseaesaaaessanessaneeses 9 As PROTOCOL Cloning Site Selection and PCR Primer Desig tt iste ues use yao iw neg de NER RWE 9 Re PROTOCOL Generating a Gene Specihe Expression EE ie sud Gae tna GU Gun LD noiis 10 VILIn Fusion Cloning Protocol u Y LAALYAAYLLAALLYSAALLAAALHAAAL SHARE HERE ARE LA REN HAE HLR LLY NL o 11 A PROTOCOL PCR Cloning2. l Introduction amp Protocol Overview Clontech Laboratories Inc ATakara Bio Company The pEcoli Expression System and pEcoli Linear Expression System are designed for the cloning and expression of his tagged proteins using E coli Figure 1 Bacterial expression is typically the first approach to the production of proteins for iv vitro studies due to its simplicity and robustness Our Bacterial Expression Systems have been designed to expedite the use of bacterial expression by incorporating a number of technologies that increase the efficiency of each of these steps This system which is based on the inducible T7 expression system pET developed by E William Studier and colleagues at Brookhaven National Laboratories Moffatt amp Studier 1986 Rosenberg et al 1987 Studier et al 1990 contains IPTG inducible pET based vectors providing high levels of protein expression Choice of cloning options The pEcoli Expression System Cat No 631417 contains two separate bacterial expression vectors encoding N or C terminal 6xHN fusion tags pEcoli Nterm 6xHN and pEcoli Cterm 6xHN respectively which provide a variety of cloning options see Figure 2 These vectors which are circular contain multiple cloning sites and can be used for traditional restriction enzyme cloning The pEcoli Expression System is available separately or as part of the EP Express and Purify Starter Kits The pEcoli Linear Expression System Cat No 631418
3. contains prelinearized versions of these vectors utilizing our In Fusion technology to provide seamless no addition of base pairs easy low background directional cloning of PCR products without the need for restriction enzyme digestion subcloning or in vitro ligation The pEcoli Linear Expression System is available separately or as part of the CEP Clone Express and Purify Starter Kits See Sections V and VI respectively for details regarding these two cloning methods Tightly regulated expression The vectors supplied with these systems contain the hybrid T7 ac promoter as well as a ac gene which encodes Lac repressor Dubendorff amp Studier 1991 This combination reduces expression in the absence of inducer while allowing for rapid inducibility upon addition of IPTG to the bacterial culture see Figure 3 This reduces background expression levels since Lac repressor binds to the ac operator present adjacent to the 17 promoter Easy purification The provided vectors are designed to incorporate 6xHN tags at the amino or carboxy terminus of the protein of in terest allowing for convenient purification using Immobilized Metal Ion Affinity Chromatography IMAC Figure 4 The 6xHN tag is short enough not to interfere with protein biological activity and function and long enough not to be internalized The EP Starter Kits which contain the pEcoli Expression System and the CEP Starter Kits which contain the pEc
4. T7 transformation polymerase under a ac regulated promoter Clones contain incorrect Pick another transformant insert Try BL21 DE3 pLysS or pLysE cells These strains con tain plasmids which express T7 lysozyme an inhibitor of T7 polymerase thus raising the threshold ofT7 poly merase required for expression of the gene of interest Protein may require Increase culture incubation time The incubation may longer induction time be performed overnight Protein is degraded Try adding protease inhibitors Confirm presence of protein by lysing cell pellet using SDS PAGE loading buffer and rescue solubility by alter ing expression conditions or expressing the protein within inclusion bodies Alter expression conditions to improve solubility by promoting correct protein folding 1 Reduce the induction temperature to slow ex pression and allow time for proper folding Try a few temperatures between 15 C and 30 C _ Refold protein in the presence of chaperones by transforming plasmids that express a variety of chaperones eg Takara Chaperone Plasmid Set Cat No 3340 or by expressing your protein in derivatives of BL21 DE3 cells that express chaperones GOETIR SAD SIO Express the protein in insoluble form within inclusion bodies then denature and refold Since insoluble proteins will typically be expressed at high levels isolation of inclusion bodies contain ing your protein of interest with one of the follow ing two m
5. expression in DEZ lysogens Tight regulation is achieved by the presence of lac operator sites in two promoters the lac UV5 promoter that controls expression of T7 RNA polymerase integrated into the genome of the DE3 lysogen and the hybrid T7 ac promoter that controls expression of the gene of interest In the absence of inducer IPTG Lac repressor expressed from both genomic and plasmid derived acl genes binds tightly to the operator sites to repress transcription When IPTG is added during induction Lac repressor releases from the operators allowing expression of T7 RNA polymerase which then acts on the newly derepressed T7 ac promoter The thick arrows refer to expression of gene products while the thin arrows indicate molecular interactions Clontech Laboratories Inc www clontech com Protocol No PT5018 1 ATakara Bio Company Version No 012512 5 pEcoli Expression Systems User Manual ll List of Components Protocol No PT5018 1 Version No 6 012512 Always store any unused In Fusion Dry Down Mix in a desiccator at 20 22 C Store all other components at 209C pEcoli Expression System Cat No 631417 10 pg 10 pg 2 ug pEcoli Nterm 6xHN Vector 500 ng ul pEcoli Cterm 6xHN Vector 500 ng ul pEcoli 6xHN GFPuv Vector 500 ng pl pEcoli Expression Systems User Manual PT5018 1 pEcoli Nterm 6xHN Vector Information Packet PT3868 5 pEcoli Cterm 6xHN Vector Information Packet PT3869 5 pEcoli 6xHN GFPu
6. of the original culture volume using PBS add an equal volume of 2X SDS loading buffer see Section III and mix well It is OK to mix samples on a vortex mixer but avoid forming bubbles if you intend to sonicate the samples 2 The samples can then be sonicated for 30 sec or drawn through a 27 gauge needle 4 or 5 times This will reduce the viscosity of the sample due to genomic DNA and thus facilitate gel loading An alternative is to centrifuge the sample for 5 min just prior to loading the gel and use the supernatant taking care to avoid the pellet when pipetting 3 Heat the sample to 95 C for 3 5 minutes centrifuge briefly and proceed to load the gel Samples may be stored at 20 C www clontech com Clontech Laboratories Inc ATakara Bio Company pEcoli Expression Systems User Manual Mil Protein Expression Protocol continued D PROTOCOL Analysis of Solubility There are numerous successful protocols for improving solubility and refolding proteins from inclusion bodies In the latter case the protein must be solubilized using denaturants prior to purification IMAC purification using TALON Protocol resin works under both native and denaturing conditions This protocol will allow you to separate soluble and insoluble inclusion body fractions and then analyze these by SDS PAGE 1 Resuspend the cell pellets in TALON xTractor Buffer a Add 20 pl TALON xTractor Buffer see Section III per mg of cell pellet or 40 u
7. e viscosity of the sample due to genomic DNA and thus facilitate gel loading An alterna tive is to centrifuge the sample for 5 min just prior to loading the gel and take care to avoid the pellet when pipetting 8 Heat the samples corresponding to both the soluble and insoluble fractions at 95 C for 3 5 min and centri fuge briefly 9 Analyze samples by SDS PAGE Add to this an equivalent volume of 2X SDS Sample Buffer Typically a sample equivalent to 25 50 ul of culture volume will produce clean bands on a gel This corresponds to 5 10 ul of sample when processed according to the above instructions 10 If you wish to identify the target protein band use our Universal HIS Western Blot Kit Cat No 635633 after transferring the bands to a PVDF membrane Clontech Laboratories Inc www clontech com Protocol No P1T5018 1 ATakara Bio Company Version No 012512 13 pEcoli Expression Systems User Manual IX Troubleshooting Guide Table I Troubleshooting Guide for Protein Expression Description of Problem Possible Explanation Solution Try BL21 DE3 pLysS or pLysE cells These strains con tain plasmids which express T7 lysozyme an inhibitor ofT7 polymerase thus raising the threshold of T7 poly merase required for expression of the gene of interest A Transformation of BL21 DE3 i Protein is toxic to cells cells yields no colonies Incorrect cells used for Use BL21 DES cells or other cells which express
8. ethods is an excellent purification step 1 Denature the proteins within the inclusion bodies in a solution of 8 M urea or 6 M guani dine hydrochloride then perform a gradual dilution of the denatured protein solution us ing stepwise dialysis Since IMAC purification works under denaturing conditions one has the option to purify either before or following refolding _ Use a commercially available refolding kit eg Takara Refolding CA Kits Cat Nos 7350 amp 7351 Protocol No PT5018 1 www clontech com Clontech Laboratories Inc Version No 012512 A Takara Bio Company 14 B Protein is not present in cells Protein is toxic to cells pEcoli Expression Systems User Manual X References Clontech Laboratories Inc ATakara Bio Company Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K Eds 2001 Current Protocols in Molecular Biology John Wiley and Sons Inc NY Dubendorff J W and Studier F W 1991 Controlling basal expression in an inducible T7 expression system by blocking the target T7 pro moter with lac repressor J Mol Biol 219 1 45 59 Moffatt B A and Studier F W 1986 Use of bacteriophage T7 RNA polymerase to direct selective high level expression of cloned genes J Mol Biol 189 1 113 130 Rosenberg A H and Studier E W 1987 T7 RNA polymerase can direct expression of influenza virus cap binding protein PB2 in Escherichia co
9. g High Fidelity PCR EcoDry Premix Cat No 639280 Transform into BL21 DE3 or other strain containing DE3 for expression analysis see Section VIII www clontech com Clontech Laboratories Inc A Takara Bio Company pEcoli Expression Systems User Manual Vil In Fusion Cloning Protocol A PROTOCOL PCR Cloning of Your PCR Insert into the pEcoli Linear Expression Vectors The following method describes a simple and highly efficient method to clone your gene in parallel into the pEcoli vectors The magic of In Fusion means Protocol e One step directional cloning Figure 5 e Seamless cloning no addition of unwanted base pairs e No restriction digestion phosphatase treatment or ligation e Clone the same PCR fragment in parallel into both expression vectors Single tube protocol Recombinant vector Figure 5 Simple one step cloning with In Fusion PCR Cloning Kits 1 Design your primers with a 15 bp homology Visit http bioinfo clontech com infusion Select In Fusion Primer Design Tool Follow the on screen directions to design your primer 2 Amplify your PCR insert by using a high fidelity enzyme such as Advantage HD Polymerase Mix Cat No 639241 3 Purify the PCR product using NucleoSpin Gel and PCR Clean Up Cat No 740609 50 With this kit you can gel purify or spin column purify the choice is yours Or Treat with Cloning Enhancer Cat No 639613 Cloning Enhancer removes background plas
10. in to design your 5 primer WITHOUT the ATG start codon of the wild type protein www clontech com Protocol No PT5019 1 Version No 012512 9 pEcoli Expression Systems User Manual Vi Protocol No PT5018 1 Version No 10 Traditional Cloning Protocol continued e Sufficient flanking sequence should be incorporated to ensure efficient digestion of the PCR product using Protocol 012512 the appropriate restriction enzymes We recommend six nucleotides 5 to the restriction enzyme recognition sequence For more information on restriction enzymes please visit TAKARA Bio USA at www takarabioUSA com am products subcategory 2 35 NOTES e The MCS is designed with overlapping Pac sites at the 3 end This ensures that all three reading frames contain a stop codon If the Pac site is used for cloning only one of the sites in the vector will be cut Thus be sure that your intended stop codon is found in the first Pac site That way the stop codon will be in frame regardless of which Pac site in the vector is digested e The Xba I site is 3 to the Pac sites and is therefore not followed by stop codons If you use the Xba site for cloning be sure that your insert contains its own stop codon B PROTOCOL Generating a Gene Specific Expression Vector Generate your recombinant expression construct using standard molecular biology techniques as described below For more detailed information see Molecula
11. l per ml of the original culture volume b Incubate at room temperature for 20 30 min the incubation with TALON x Tractor buffer can be carried out on ice for 45 60 min MI NOTE Add DNAse to TALON xTractor Buffer if the resulting sample is very viscous 2 Alternatively resuspend the cell pellets in 1X Equilibration Wash Buffer as follows a Resuspend the cell pellets in a volume of the provided 1X Equilibration Wash Buffer equivalent to 1 10th the original culture volume chilled to 47C b Add lysozyme to a concentration of 0 75 mg ml To reduce the chance of introducing proteases use the highest purity lysozyme available c Sonicate your sample 3 x 10 sec with a 30 sec pause on ice between each burst Centrifuge the cell extract at 10 000 12 000 x g for 20 min at 47C to pellet insoluble material Carefully transfer the supernatant to a clean tube without disturbing the pellet This is the clarified sample 5 Reserve a portion for SDS PAGE analysis The remainder may be kept for a trial purification using TALON resin Keep the remainder on ice until it is used for purification carry out the purification within 2 4 hr after the extraction 6 Resuspend the pellet in a volume of 1X Equilibration Wash Buffer equivalent to the volume used in Step 1 Add an equal volume of 2X SDS sample buffer 7 he resuspended pellet can then be sonicated for 30 sec or drawn through a 27 gauge needle 4 or 5 times This will reduce th
12. li Gene 59 2 3 191 200 Sambrook J amp Russell D W 2001 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor NY Studier E W Rosenberg A H Dunn J J and Dubendorff J W 1990 Use of T7 RNA polymerase to direct expression of cloned genes Methods Enzymol 185 60 89 Notice to Purchaser Clontech products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro diagnostic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufacture commercial products or to provide a service to third parties without prior written approval of Clontech Laboratories Inc Your use of this product is also subject to compliance with any applicable licensing requirements described on the product s web page at http www clontech com It is your responsibility to review understand and adhere to any restrictions imposed by such statements Clontech the Clontech logo Advantage EcoDry HisTALON In Fusion Stellar and TALON are trademarks of Clontech Laboratories Inc All other marks are the property of their respective owners Certain trademarks may not be registered in all jurisdictions Clontech is a Takara Bio Company 2012 Clontech Laboratories Inc This document has been reviewed and approved by the Clontech Quality Assurance Depar
13. mid DNA and PCR residue eliminating the need for PCR insert purification 4 Clone Combine your PCR insert and linearized vectors with the In Fusion Enzyme and incubate The same PCR insert can be cloned into both C term and N term vectors 5 Transform and screen colonies for the correct insert See the In Fusion Dry Down PCR Cloning Kit User Manual PT3754 1 at www clontech com for additional details regarding the PCR cloning procedure Clontech Laboratories Inc www clontech com Protocol No P1T5018 1 ATakara Bio Company Version No 012512 11 pEcoli Expression Systems User Manual Mil Protein Expression Protocol Protocol Protocol Protocol No PT5018 1 Version No 012512 12 A General Considerations Once the desired expression plasmid has been obtained you may induce expression of your protein using IPTG according to the following recommendations e First the plasmid must be transformed into E coli strain BL21 DE3 EMD Biosciences Inc which contains the T7 polymerase gene under the control of the ac repressor e It is recommended to use freshly transformed bacteria or frozen glycerol stocks for expression experiments since continued culture or long periods of storage on plates can result in loss of expression The following protocols are provided for the analysis of protein expression including protein solubility This small scale approach is also useful for optimizing expression conditions The exp
14. n E coli the expressed N or C terminal tagged protein is efficiently purified using either HisTALON resin for highest purity of the target protein or His60 Ni Superflow resin for the highest binding capacity N terminal vector Sall Hind Ill Stu Eco RI Pst Bgl ll Not Nco I Pac T7 lac Enterokinase Removed in promoter cleavage site prelinearized In Fusion Ready vectors C terminal vector Sall Bindi Not Stu Eco RI e Nco Doll Pst Pac T7 lac Removed in t Thrombin promoter prelinearized In Fusion cleavage site Ready vectors Figure 2 Cloning options using the pEcoli Expression System Features of the multiple cloning sites of our pEcoli Expression Vectors are shown not to scale Transcription proceeds from left to right beginning at ATG and ending at the stop TAA codon shown Convenient restriction sites indicated allow inclusion or removal of tags as needed The unshaded region is removed in our pEcoli Linear Expression Vectors using Sal and Hind Ill restriction enzymes www clontech com Clontech Laboratories Inc ATakara Bio Company pEcoli Expression Systems User Manual I Introduction amp Protocol Overview continued Host genome DEZ lysogen wu laclgene vy B sse Lac repressor Ma fA wu lac promoter T7 gene 1 wv DEZ L Protein f A S of interest K pEcoli vector T7 RNA polymerase Figure 3 The molecular basis of the pET system for recombinant protein
15. of Your PCR Insert into the pEcoli Linear Expression Vectors 11 MILProtein Expression Protocol ccccccsccceeeeeceeeeeeeeeecaeesaeeeeesaesaeseeneeeesaneseaseeesaneseessensanesaness 12 Pi EC EE 12 Be PROTOCOL Expression Protocol mall 5c Ale e geesde eegenen gege 12 Eeer Express ES eege 12 IER NEE EE EE 13 IX Troubleshooting CTT EB 14 A ROIENC OS ooa E O ne dete E E S EGO 15 List of Figures Figure 1 The BP and BP Starter Kits protocol ssisiseiitesnisicarasn deneden e OR 4 Figure 2 Cloning options using the pEcoli Expression System ssssssssesesssessssseseresssssereserssersssressesserssns 4 Figure 3 The molecular basis of the pET system for recombinant protein expression in DE3 lysogens 5 Figure 4 Bacterial expression and purification flowchart ice sesnasesaveceeancaadzcueseenanseedcseysalacnenatedectnasetadanteute 9 Figure 5 Simple one step cloning with In Fusion PCR Cloning Kits 0 0 ees eeeceeeseeeeeseeeeeeeeeeeeeeneeens 11 List of Tables Table I Troubleshooting ER 14 Customer Service Ordering Technical Support tel 800 662 2566 toll free tel 800 662 2566 toll free fax 800 424 1350 toll free fax 650 424 1064 web www clontech com e mail tech clontech com e mail orders clontech com Protocol No PT5018 1 www clontech com Clontech Laboratories Inc Version No 012512 A Takara Bio Company 2 pEcoli Expression Systems User Manual
16. oli Linear Expression System come with your choice of purification resin and buffers to perform extraction and purification of his tagged proteins Choose His T ALON Co resin to achieve the highest purity of your target protein or choose His60 Ni Superflow resin when a high binding capacity is required www clontech com Protocol No PI5018 1 Version No 012512 3 pEcoli Expression Systems User Manual I Introduction amp Protocol Overview continued Protocol No PT5018 1 Version No 4 012512 EP Starter Kits CEP Starter Kits pEcoli Expression System Pm pEcoli Linear Expression System We Gene of Sek interest Cloning of restriction fragments In Fusion cloning of PCR products into circular pEcoli vector into prelinearized pEcoli vector using T4 DNA Ligase l N or C terminal N or C terminal 6xHN tagged construct 6xHN tagged construct Transform construct into E coli and express protein HisTALON purified protein His60 Ni purified protein with N or C terminal with N or C terminal 6xHN tag 6xHN tag Figure 1 The EP and CEP Starter Kits protocol A gene of interest is cloned into a pEcoli vector a pET based expression system to generate an N or C terminal tagged construct The pEcoli Expression System allows a choice of cloning sites while the pEcoli Linear Expression System provides the option of easy precise In Fusion PCR cloning to yield a 6xHN tagged construct After transformation and growth i
17. r Cloning A Laboratory Manual Sambrook amp Russell 2001 or Current Protocols in Molecular Biology Ausubel er al 2001 1 Digest the pEcoli vector with the restriction enzyme s appropriate for your expression application treat with phosphatase if desired and purify NOTES e The ends of the fragment to be cloned must be compatible with one or more restriction sites present in the cloning vector This can be accomplished by PCR amplifying the fragment using primers that contain suit able restriction sites e f cloning with the 6xHN affinity tag included in the vector ensure that the correct reading frame is main tained see the MCS of the appropriate vector for the alignment of restriction sites with the open reading frame that begins with the initiation codon ATG within the Nco I site Purify the insert using any standard method We recommend using the NucleoSpin Gel and PCR Clean Up Cat No 740609 50 This kit can be used for PCR clean up or gel purification Ligate the digested vector and the gene fragment Transform chemically competent or electrocompetent bacterial cells we recommend Clontech s Stellar Electrocompetent Cells Cat No 636765 with a sample of the ligation mixture and plate on LB agar plates containing 100 pg ml ampicillin Identify the desired recombinant plasmid by colony PCR restriction analysis of miniprepped DNA and or sequencing of miniprepped DNA For colony PCR we recommend usin
18. ression protocol can be scaled up for production and IMAC purification can be performed using either HisTALON Co resin for the highest purity or His60 Ni Superflow resin for the highest binding capacity B PROTOCOL Expression Protocol Small Scale 1 Pick colonies to innoculate small 2 4 ml LB amp cultures and shake overnight at 37 C ampicillin concen tration is 100 pg ml In the morning innoculate a 4 50 ml culture by diluting the overnight culture 1 20 to 1 50 with LB amp 2 Shake until an OD of 0 6 0 8 is reached set aside a small volume of culture 1 ml on ice to serve as an uninduced control and then add IPTG to a concentration of 1 mM Continue shaking at 37 C for 4 5 hr 3 Centrifuge samples at 1 000 3 000 x g for 15 min at 47C remove supernatant and freeze pellets at 80 C for further analysis C PROTOCOL Analysis of Expression You have the option to analyze total protein expression when analyzing a number of samples or proceed directly to analyzing solubility see Section D Protein solubility is essential when planning the purification protocol since some eukaryotic proteins are insoluble when expressed in bacteria and form inclusion bodies The trade off of insolubility is that these proteins tend to be expressed at high levels and isolation of inclusion bodies is an effective initial purification step Total expression can be analyzed by SDS PAGE as follows 1 Resuspend pellets in 1 10th
19. ssolve 238 mg of IPTG in 10 ml of deionized H O Filter sterilize and store in 1 ml aliquots at 20 C www clontech com Protocol No PT5018 1 Version No 012512 7 pEcoli Expression Systems User Manual IV Workflow Transform into Expression Host e Transform into BL21 DE3 cy competent bacteria EMD Biosciences Inc Protocol No PT5018 1 www clontech com Clontech Laboratories Inc Version No 012512 A Takara Bio Company 8 Figure 4 Bacterial expression and purification flowchart pEcoli Expression Systems User Manual V General Considerations Traditional Cloning Method Choose the pEcoli Expression System Cat No 631417 or any of the EP Starter Kits Cat Nos 631419 631420 631421 631416 amp 631426 which contain circular vectors for traditional restriction enzyme based cloning Using traditional cloning methods De ligation based cloning of compatible DNA fragments provides a choice of several cloning sites Figure 2 provided that these sites are not found internally in the DNA fragment to be cloned Traditional cloning may be more convenient if the fragment to be cloned contains restriction sites in frame with the gene of interest For detailed vector maps please see the Vector Information Packets listed in Section HI PCR Cloning Method Choose the pEcoli Linear Expression System Cat No 631418 or any of the CEP Starter Kits Cat Nos 631422 631423 631424 631425 amp 631427
20. system depending on your needs Vector Maps The vector maps and multiple cloning site information for each of the pEcoli system vectors are provided in the Vector Information Packets listed in Section II which are available at www clontech com VI Traditional Cloning Protocol Protocol Clontech Laboratories Inc ATakara Bio Company PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING Traditional cloning consists of many steps These include primer design PCR amplification and cleanup of the PCR insert or cleavage from an existing vector and insert cleanup The vector must also be cleaved and cleaned up This is followed by T4 DNA ligation transforma tion and screening of inserts We recommend the DNA Ligation Kit Ver 1 0 TAKARA Bio USA Cat No TAK 6021 or the DNA Ligation Kit Ver 2 1 TAKARA Bio USA Cat No TAK 6022 A PROTOCOL Cloning Site Selection and PCR Primer Design Use the following criteria to select restriction sites for cloning and to design PCR primers for generating the DNA insert e Choose appropriate restriction sites based on the absence of these sites within the DNA insert to be cloned e These sites should be incorporated into PCR primers which are then used to PCR amplify the sequence to be cloned e These primers should be carefully designed to ensure that the reading frame of your gene aligns with the ATG start codon which forms part of the Noo I site at the 5 end of the MCS e Make certa
21. tive clones following colony screening TALON xTractor Buffer Cat No 635656 for solubility analysis and extraction of expressed proteins ProteoGuard EDTA Free Protease Inhibitor Cocktail Cat Nos 635672 or 635673 5X SDS loading buffer 15 B mercaptoethanol 15 SDS 50 glycerol 1 5 bromophenol blue Stellar Electrocompetent Cells Cat No 636765 BL21 DE3 competent bacteria EMD Biosciences Inc ampicillin 100 mg ml stock LB Luria Bertani medium pH 7 0 for 1 L 1 0 Bacto tryptone 10 g 0 5 yeast extract 5 g 1 0 NaCl 10 g Dissolve ingredients in 950 ml of deionized H O Adjust the pH to 7 0 with 5 M NaOH and bring the volume up to 1 L Autoclave on liquid cycle for 20 min at 15 lb in Store at room temperature or at 47C LB antibiotic plates Prepare LB medium as above but add 15 g L of agar before autoclaving Autoclave on liguid cycle for 20 min at 15 lb in Let cool to 55 C add antibiotic e g 100 pg ml of ampicillin and pour into 10 cm plates After the plates harden then invert and store at 4 C For LB X Gal IPTG plates spread 40 ul each of X Gal and IPTG stock solutions on an LB plate X Gal stock solution 5 bromo 4 chloro 3 indolyl B D galactoside 40 mg ml in dimethylformamide Dissolve 400 mg of X Gal in 10 ml of dimethylformamide Protect from light by storing in a brown bottle at 20 C IPTG stock solution isopropyl B D thiogalactoside 100 mM Di
22. tment www clontech com Protocol No PT5018 1 Version No 012512 15
23. v Vector Information Packet PT3870 5 pEcoli Linear Expression System Cat No 631418 9 rxns 1 5 ug 1 5 ug gt ug 3 ul In Fusion Dry Down Mix 1 x 8 well strip pEcoli Nterm 6xHN Linear Vector 100 ng ul pEcoli Cterm 6xHN Linear Vector 100 ng ul pEcoli 6xHN GFPuv Vector 500 ng pl 1 1 kb LacZ RK Control Insert 25 ng ul pEcoli Expression Systems User Manual PT5018 1 pEcoli Nterm 6xHN Linear In Fusion Ready Vector Information Packet PT3871 5 pEcoli Cterm 6xHN Linear In Fusion Ready Vector Information Packet P13872 5 pEcoli 6xHN GFPuv Vector Information Packet PT3870 5 www clontech com Clontech Laboratories Inc ATakara Bio Company pEcoli Expression Systems User Manual lil Additional Materials Required The following materials are required but not supplied Clontech Laboratories Inc ATakara Bio Company Advantage HD Polymerase Mix Cat Nos 639241 for amplifying your gene of interest NucleoSpin Gel and PCR Clean Up Cat No 740609 50 for purification of vector and insert if using pEcoli Expression System or purification of insert if using pEcoli Linear Expression System DNA Ligation Kit Ver 1 0 TAKARA Bio USA Cat No TAK 6021 or DNA Ligation Kit Ver 2 1 TAKARA Bio USA Cat No TAK 6022 High Fidelity PCR EcoDry Premix Cat No 639280 for colony PCR screening NucleoSpin QuickPure Cat No 740615 50 or NucleoSpin Plasmid Cat No 740588 50 for purification of posi
24. which contain prelinearized vectors designed to be used with Clontech s In Fusion technology In Fusion cloning provides several advantages over restriction enzyme based cloning e Directional cloning of PCR products into linearized vectors without the need for restriction digests ligation or blunt end polishing This is more convenient than restriction enzyme based cloning and allows the use of restriction sites that are present within the fragment to be cloned e No restriction digestion Since the vectors are provided in prelinearized form using Sal I and Hind III there is no need to digest the vector prior to cloning In Fusion cloning is based on homology between the ends of the linearized vector and the PCR product this homology is introduced into the PCR producc via the use of PCR primers with 5 extensions e Clone the same PCR product into both vectors in parallel The pEcoli Nterm 6xHN and pEcoli Cterm 6xHN vectors have been designed so that the sequences 5 to the Sal I site and 3 to the Hind III site are the same in both vectors Ihis allows cloning of the same PCR product into both vectors in parallel which is convenient for comparing expression and purification of your protein when a 6xHN tag is attached to either the amino or the carboxy terminus respectively Furthermore these PCR products are compatible with all of our prelinearized fluorescent protein vectors allowing you to easily switch to a different expression
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