Cloning into pTrcHis A, B, and C
Contents
1. 1 M glucose 100 mM IPTG 10 For 100 ml Dissolve 19 grams of MgCl in deionized water Bring the final volume to 100 ml and filter sterilize 0 22 micron filter For 100 ml Dissolve 18 grams of glucose in 90 ml of deionized water Bring the final volume to 100 ml and filter sterilize 0 22 micron filter For 10 ml of a 100 mM solution Dissolve 0 24 g of IPTG m w 238 3 in 10 ml of sterile deionized water Filter sterilize and store at 20 C Map of pTrcHis A B and C Vectors pTrcHis A B and C The figure below summarizes the features of the pTrcHis vectors Details of the Map multiple cloning site are shown on pages 3 4 The full sequence of pTrcHis is available for downloading from our web site at www invitrogen com or by contacting Technical Support see page 14 BamH Xho l Ava C Nhe Xpress Epitope ATG 6xHis EK MCS pIrcHis A B C Sph 1 Comments for pTrcHis B 4404 nucleotides trc promoter bases 191 221 lac operator bases 228 248 rrnB anti termination sequences bases 264 333 T7 gene 10 translational enhancer bases 346 354 Ribosome binding site bases 370 374 Mini cistron bases 383 409 Polyhistidine and enterokinase cleavage site bases 425 504 Xpress epitope bases 482 505 Multiple cloning site bases 515 554 rrnB transcriptional termination sequence bases 637 794 Ampicillin resistance ORF bases 1074 1934 pBR322 origin bases 202. or technical assistance call write fax or email Additional international offices are listed on our web site www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS MSDSs Material Safety Data Sheets are available on our web site at Certificate of Analysis Limited Warranty 14 www invitrogen com msds Product qualification is described in the Certificate of Analysis CofA available on our website by product lot number at www invitrogen com support Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free o
3. 10 Chemically Competent E coli 10 x 50 ul C4040 10 vi Overview Introduction Detection and Purification of Recombinant Protein Introduction The pTrcHis vectors are pBR322 derived expression vectors designed for efficient recombinant protein expression and purification in E coli High levels of expression are possible using the tre trp lac promoter Egon et al 1983 and the rrnB anti termination region Li et al 1984 The trc promoter contains the 35 region of the trp promoter together with the 10 region of the lac promoter Brosius et al 1985 Egon et al 1983 Mulligan et al 1985 The pTrcHis vectors also contain a copy of the Jacl gene which codes for the lac repressor protein This allows for efficient repression of transcription of the cloned insert in E coli regardless of whether the strain is lacI or lacI When expression is desired the E coli are grown to mid log phase and IPTG isopropyl B D thiogalactoside is added to 1 mM to induce expression via derepression Translation is enhanced by the presence of a minicistron that provides highly efficient translational restart into the open reading frame ORF of the multiple cloning site MCS DNA inserts are positioned downstream and in frame with a sequence that encodes an N terminal fusion peptide The N terminal peptide codes for 5 to 3 from the promoter an ATG translation initiation codon six histidine residues in ser
4. 5 ml of 100 mM IPTG stock to 50 ml of culture and grow at 37 C with vigorous shaking for the optimal time determined as described on the previous page Proceed with protein purification as detailed in the ProBond Purification System Manual The ProBond Purification System Manual is available for downloading from our web site at www invitrogen com or by contacting Technical Support see page 14 Appendix Transformation Protocol for Competent E coli Protocol Preparing Frozen E coli Glycerol Stocks The following protocol is provided for your convenience Any comparable protocol may be used 1 Take the TOP10 stab provided and streak out a small portion of it on an LB media plate without ampicillin Incubate at 37 C overnight Store the stab at 4 C in the dark it should be viable for several months Important Prepare a frozen glycerol stock for long term storage see below for instructions Pick a single colony and transfer it into 100 ml of SOB media in a 1 liter flask see page 9 for recipe Incubate the flask at 37 C with vigorous shaking gt 200 cycles minute in a rotary shaker When the OD reaches approximately 0 5 collect the cells by centrifuging at 2 600 x g for 10 minutes at 4 C Resuspend the pellet in 10 ml of ice cold 50 mM CaCh Keep the cells on ice for at least 30 minutes Centrifuge the CaCl2 treated cells in a 4 C rotor 2 600 x g 4 C 5 minutes Gently resuspend the cell
5. 79 2752 lac I4 ORF bases 3406 4365 11 Features of pTrcHis Vector Features of pTrcHis 12 The important elements of pTrcHis A B and C are described in the table below All features have been functionally tested Feature Benefit trc promoter 35 trpB and 10 lacUV5 hybrid promoter for high level expression of fusion protein Brosius et al 1985 Egon et al 1983 Mulligan et al 1985 lac operator Permits binding of the Lac repressor to repress transcription rrnB anti termination sequences Reduces the level of premature transcription termination Li et al 1984 Bacteriophage gene 10 translational enhancer Optimizes translation initiation of minicistron Olins et al 1988 Minicistron and reinitiation ribosome binding site Contains a second ribosome site for efficient reinitiation of translation into the gene of interest Schoner et al 1986 Polyhistidine 6xHis region Permits purification of recombinant fusion protein on metal chelating resins i e ProBond Enterokinase cleavage site Provides a site for efficient removal of the fusion tag Multiple cloning site Allows insertion of your gene for expression rrnB transcription terminator Strong transcription termination region Ampicillin resistance gene B lactamase Allows selection of the plasmid in E coli pBR322 origin Low copy replication and growth in E co
6. AAATTAAAG AGGTATATAT TA ATG TAT CGA TTA AAT AAG GAG GAA TAA ACC Met Tyr Arg Leu Asn Lys Glu Glu Polyhistidine 6xHis region lr re ATG GGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT AGC ATG ACT Met Gly Gly Ser His His His His His His Gly Met Ala Ser Met Thr Xpress epitope EK recognition sequence HAT EK cleavage site GGT GGA CAG CAA ATG GGT CGG ACT CTG TAC GAC GAT GAC GAT AAG GAT Gly Gly Gln Gln Met Gly Arg Thr Leu Tyr Asp Asp Asp Asp Lys Asp BamH Xho Sac I Bgl II I I I AGA TGG GGA TCC GAG CTC GAG ATC TGC Arg Trp Gly Ser Glu Leu Glu Ile Cys Hind Hi CGA AGC TTG GCT GTT TTG GCG GAT GAG Arg Ser Leu Ala Val Leu Ala Asp Glu Pst AGC TGG Ser Trp AGA AGA Arg Arg Kpn I TAC CAT Tyr His TTT TCA Phe Ser EcoR BstB ATG GGA ATT Met Gly Ile GCC TGA Ala Continued on next page Cloning into pTrcHis A B and C Continued Multiple Cloning Site of pTrcHis B 361 413 ATG Met 461 GGT Gly Below is the multiple cloning site for pTrcHis B Restriction sites are labeled to indicate the actual cleavage site Boxed nucleotides indicate the variable region Sequencing and functional testing have confirmed the multiple cloning site The complete sequence of pTrcHis B is available for downloading from our web site at www invitrogen com or by contacting Technical Support page 14 For a map and description of the features of pTrcHis refer to pages 11 12 pTrcHis fo
7. DNA into the MCS in frame with the ATG To perform in frame cloning three different versions of the vector pTrcHis A pTrcHis B and pTrcHis C that differ only in the spacing between the sequences that code for the N terminal peptide and the MCS are supplied For proper expression first determine which restriction site is appropriate for ligation and then which vector will preserve the reading frame between the 5 sequences and the insert when ligated into that site This will vary depending on which restriction site in the MCS is chosen for fragment insertion not all cloning sites are in the same frame in each vector The complete sequences for the pTrcHis vectors are available for downloading from our web site at www invitrogen com or by contacting Technical Support see page 14 Continued on next page Cloning into pTrcHis A B and C Continued Multiple Cloning Site of pTrcHis A 361 413 461 509 557 Below is the multiple cloning site for pTrcHis A Restriction sites are labeled to indicate the actual cleavage site Boxed nucleotides indicate the variable region Sequencing and functional testing have confirmed the multiple cloning site The complete sequence of pTrcHis A is available for downloading from our web site at www invitrogen com or by contacting Technical Support page 14 For a map and description of the features of pTrcHis refer to pages 11 12 pTrcHis Forward Primer RBS Mini cistron A
8. Invitrogen pTrcHis A B and C Vectors for Expression of Recombinant Proteins Containing N Terminal 6xHis Tags in E coli Catalog no V360 20 Version H 20 October 2008 25 0038 Table of Contents Kit Contents and Storage nnnnnsnnenenranenenenenenenenenensenenenenenenenenenenenenenenenseeenenenenenensenenenenenennnenenenenenenenens v Accessory Productserie ban e debit eite ata vi Introduction EARE E T E E E T 1 OA A Lunn sek kai den oie ede de kerk A A nie d rede iu bn d ia 1 Methods 2 Cloning into pTrcHis A B and C eene eas SRA tenente tenens 2 O oai dani dentes deert 5 Ppo 7 Transformation Protocol for Competent E coli nnn ennen eenen nrenenenenenenenenenenenenenenenevenenenenenenenenenenee 7 Bacterial Alkaline Lysis Miniprep nnnsananenenenenenenenereeneneneneneneenenenenenennneneneneneneneneneneenenenenenenensnenenne 8 O dae 9 Map of pTrcHis A B and C Vectors nnnanannenenseneneneneneaneneneneneneneneneneenenenenenenenenenenenensenenenenenenenenenn 11 Features Of PIreHis Vector gan eine eee n epe deir engl lave teer sa eie dette es 12 Map of pIrcEls CAT ette NEO 13 Technical Support wader lana PORUM Detto ia 14 Putchaser Notification cete entere n de ie ie TRE Pee to ere e nete eiii terii ferens 15 References ai 16 111 iv Kit Contents and Storage Shipping a
9. Ltd Basel Switzerland and is provided only for use in research Information about licenses for commercial use is available from QIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany 15 References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience New York Brosius J Erfle M and Storella J 1985 Spacing of the 10 and 35 Regions in the tac Promoter J Biol Chem 260 3539 3541 Egon A Brosius J and Ptashne M 1983 Vectors Bearing a Hybrid trp lac Promoter Useful for Regulated Expression of Cloned Genes in Escherichia coli Gene 25 167 178 Li S C Squires C L and Squires C 1984 Antitermination of E coli rRNA Transcription is Caused by a Control Region Segment Containing Lambda nut like Sequences Cell 38 851 860 Mulligan M E Brosius J and Clure W R 1985 Characterization in vitro of the Effect of Spacer Length on the Activity of Escherichia coli RNA Polymerase at the tac Promoter J Biol Chem 260 3539 3538 Olins P O Devine C S Rangwala S H and Kavka K S 1988 T7 Phage Gene 10 Leader RNA a Ribosome binding Site the Dramatically Enhances the Expression of Foreign Genes in Escherichia coli Gene 73 227 235 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Ed Cold Spring H
10. arbor Laboratory Press Plainview New York Schoner B E Belagaje R M and Schoner R G 1986 Translation of a Synthetic Two cistron mRNA in Escherichia coli Proc Natl Acad Sci USA 83 8506 8510 2001 2008 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 16 invitrogen Corporate Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
11. atant sample add an equal volume of 2X Laemmli Buffer Analyze 10 20 ul of both the supernatant and pellet samples on a 10 SDS polyacrylamide gel Stain the gel with Coomassie blue and look for a band of increasing intensity in the expected size range for the protein Compare it to the negative control time course to distinguish the recombinant proteins from the background proteins 10 Determine the optimal time post IPTG induction to harvest the cells The N terminal fusion peptide adds approximately 3 kDa to the size of your protein Continued on next page Expression Continued Positive Control Included is a stab of E coli strain TOP10 containing pTrcHis CAT Vector pTrcHis CAT contains the chloramphenicol acetyl transferase gene CAT for use as a positive control for expression The CAT protein is approximately 30 kD It begins to appear at approximately 0 5 hours after IPTG induction and reaches a peak level of expression after approximately 4 hours post induction At that point expression levels off and continues for several hours Expression Of 1 Inoculate 2 ml of SOB Ampicillin 50 ug ml with a single recombinant Recombinant E coli colony Grow overnight at 37 C with shaking Protein 2 The next day inoculate 50 ml of SOB Ampicillin 50 ug ml with 0 3 ml of y P 8 the overnight culture Grow the culture at 37 C with vigorous shaking to an ODso 0 6 Add IPTG to a final concentration of 1 mM 0
12. ector to transform a recA endA E coli strain like TOP10 DH5a or equivalent Transformants are selected on LB plates containing 50 100 ug ml ampicillin The E coli strain TOP10 is provided for propagation of the pTrcHis vector E coli strains with comparable genotypes may be substituted We recommend that you propagate vectors containing inserts in recombination deficient recA endonuclease A deficient endA E coli strains TOP10 contains e recA for stable replication of high copy number plasmids e endA for improved yield and quality of miniprep DNA e hsdRMS to eliminate cleavage of recombinant plasmid by the endogenous EcoR restriction system e For your convenience TOP10 is available as both electrocompetent and chemically competent cells from Invitrogen page vi F mcrA A mrr hsdRMS mcrBC 80lacZAM15 AlacX74 recA1 araD139 ara leu 7697 galU galK rpsL endA1 nupG You may use any method of choice for transformation Chemical transformation is the most convenient for many researchers Electroporation is the most efficient and the method of choice for large plasmids Refer to Appendix page 7 for protocols Downstream of the 5 sequences there is a multiple cloning site MCS that has eight unique restriction sites BamH I Xho I Bel IL Pst I Kpn I EcoRI BstB I and Hind III refer to pages 3 4 for details To generate recombinant proteins that include the correct N terminal fusion peptide clone the
13. f charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Purchaser Notification Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer This product is licensed under U S Patent Nos 5 284 933 and 5 310 663 and foreign equivalents from Hoffmann LaRoche Inc Nutley NJ and or Hoffmann LaRoche
14. for downloading from our web site at www invitrogen com or by contacting Technical Support page 14 Fora map and description of the features of pTrcHis refer to pages 11 12 TrcHis forward primer Mini cistron RBS I 1 ATG TAT CGA TTA AAT AAG GAG GAA TAA ACC Met Tyr Arg Leu Asn Lys Glu Glu Polyhistidine 6x His region CAT CAT CAT CAT CAT CAT GGT ATG GCT AGC ATG ACT His His His His His His Gly Met Ala Ser Met Thr Xpress Epitope EK recognition sequence Y EK cleavage site GAT CTG TAC GAC GAT GAC GAT AAG CAT Asp Leu Tyr Asp Asp Asp Asp Lys His TCT Ser CAA Gln ATG Met Xho I I GGT CGG Gly Arg Bgl Il I 509 dea TGG ATC Pst I Kpn 1 EcoR I BstB I I I CGA CTG CAG CTG GTA CCA TAT GGG AAT TCG CCT CGA GAT Arg Hind UI I 557 AAG Lys CTT GGC Leu Gly rp Ile Arg Pro Arg Asp Leu Gln Leu Val Pro Tyr Gly Asn Ser TGT Cys TTT Phe GGC GGA Gly Gly TGA GAG AAG ATT TTC k k AGC CTG ATA CAG Expression Introduction Pilot Expression Note Since each recombinant protein has different characteristics that may affect optimal expression parameters we recommend performing a time course of expression to determine the optimal expression conditions for your particular protein Be sure to perform mock expression consisting of pTrcHis vector alone in parallel as a negative control Use the pIrcHisCAT as a positive expression co
15. g Once autoclaved add 5 ml of sterile 2 M MgCl alternatively use 10 ml of either sterile 1 M MgCl or sterile I M MgSO Follow recipe as per SOB After autoclaving let cool to about 60 C or less comfortable to touch with hand and add 20 ml of a 1 M solution of glucose Mix the media well Component liquid plates top agar Tryptone 10g 10g 10g Yeast Extract 5g 5g 5g NaCl 10 g 10 g 10 g Agar msme 15g 7g 1 Combine the tryptone yeast extract and NaCl with 950 ml of deionized water Mix the solution until dissolved 2 Adjust the pH to 7 0 with 5 N NaOH will take about 0 2 ml For plates add the appropriate amount of agar after adjusting the pH Adjust volume to 1 liter with water 4 Sterilize by autoclaving Prepare a stock solution of 50 mg ml in deionized water Filter sterilize through a 0 22 micron filter To prepare media containing ampicillin cool media to 50 C add 1 ml of the ampicillin stock per liter of media both liquid and solid for a final concentration of 50 ug ml Store the stock solution at 20 C For 100 ml of a 50 mM solution Dissolve 0 56 g of anhydrous CaCl in 100 ml of deionized water Filter sterilize 0 22 micron filter Use this solution ice cold for competent cell preparation For 100 ml Dissolve 1 86 grams KCI in deionized water Bring the final volume to 100 ml and filter sterilize 0 22 micron filter Continued on next page Recipes Continued 2M MgCl
16. ies that function as a metal binding domain in the translated protein the bacteriophage T7 gene 10 translation enhancer the Xpress epitope and an enterokinase cleavage recognition sequence Expression of your recombinant protein can be detected using an antibody to the Xpress epitope encoded in the N terminal fusion peptide i e Anti Xpress Antibody In addition the metal binding domain of the fusion peptide allows simple one step purification of recombinant proteins by Immobilized Metal Affinity Chromatography IMAC using Invitrogen s ProBond resin page vi The enterokinase cleavage recognition site in the fusion peptide between the metal binding domain and the recombinant protein allows for subsequent removal of this N terminal fusion peptide from the purified recombinant protein Methods Cloning into pTrcHis A B and C General Molecular Biology Techniques Maintenance of pTrcHis E coli Strain Genotype of TOP10 Transformation Method Cloning into the Expression Vector The following information is provided to help you clone your gene of interest into pTrcHis For help with DNA ligations E coli transformations restriction enzyme analysis DNA sequencing and DNA biochemistry see Molecular Cloning A Laboratory Manual Sambrook et al 1989 or Current Protocols in Molecular Biology Ausubel et al 1994 If you wish to propagate and maintain pTrcHis we recommend using 10 ng of the v
17. li laci gene Encodes and overproduces the Lac repressor protein Map of pTrcHis CAT pTrcHis CAT Map The figure below summarizes the features of the pIrcHis CAT vector The complete nucleotide sequence for pTrcHis2 CAT is available for downloading from our web site at www invitrogen com or by contacting Technical Support page 14 ATG 6xHis XPress EK CAT Comments for pTrcHis CAT 5191 nucleotides trc promoter bases 191 221 lac operator bases 228 248 rrnB antitermination sequences bases 264 333 T7 gene 10 translational enhancer bases 346 354 Ribosome binding site bases 370 374 Mini cistron bases 383 409 Initiation ATG bases 413 415 Polyhistidine 6xHis region 425 442 Xpress epitope bases 482 505 Enterokinase EK recognition site bases 491 505 CAT ORF bases 587 1246 rrnB transcriptional termination sequence bases 1424 1581 Ampicillin resistance ORF bases 1861 2721 pBR322 origin bases 2866 3539 lac Id ORF bases 4068 5152 13 Technical Support Web Resources Visit the Invitrogen web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information
18. nd Storage Kit Contents The vectors included with Catalog no V360 20 are shipped on wet ice Upon receipt store vectors at 20 C Upon receipt store stabs at 4 C The following components are included with Catalog no V360 20 Note that the vectors are supplied in suspension Note For long term storage of your stab strains we recommend preparing a glycerol stock immediately upon receipt and storing at 80 C Component Quantity Composition pIrcHis A Expression Vector 20 ug 40 ul of 0 5 ug ul plasmid DNA in 10 mM Tris HCl 1 mM EDTA pH 8 0 pIrcHis B Expression Vector 20 ug 40 ul of 0 5 ug ul plasmid DNA in 10 mM Tris HCl 1 mM EDTA pH 8 0 pTrcHis C Expression Vector 20 ug 40 ul of 0 5 ug ul plasmid DNA in 10 mM Tris HCl 1 mM EDTA pH 8 0 TOP10 stab pTrcHis CAT 1 Stab m Positive Control TOP10 E coli stab 1 Stab x Accessory Products Additional The following products are available separately from Invitrogen For more Products information refer to our web site at www invitrogen com or contact Technical Support page 14 Product Quantity Catalog No Anti Xpress Antibody 50 pl R910 25 Anti Xpress HRP Antibody 50 pl R911 25 ProBond Resin 50 ml R801 01 150 ml R801 50 ProBond Purification System 6 purifications K850 01 EKMax 250 units E180 01 One Shot Top 10 Electrocomp E coli 10 x 50 ul C4040 50 One Shot Top
19. ntrol Inoculate 2 ml of SOB or LB Ampicillin 50 ug ml with a single recombinant E coli colony Grow overnight at 37 C with shaking The next day inoculate 50 ml of SOB or LB Ampicillin 50 ug ml with 0 2 ml of the overnight culture Grow the culture at 37 C with shaking to an OD600 0 6 the cells should be in mid log phase Remove a 1 ml aliquot of cells prior to IPTG induction centrifuge the sample in a microcentrifuge aspirate the supernatant Freeze at 20 C This will be the time zero sample Add IPTG to a final concentration of 1 mM 0 5 ml of 100 mM IPTG stock to 50 ml of culture and grow at 37 C with shaking Take samples at one hour intervals for 5 hours or more Centrifuge each sample and store both the supernatant and the pellet at 4 C For long term storage gt 5 hours store the samples at 20 C When all time points are collected resuspend each pellet in 100 ul of 20 mM phosphate buffer at neutral pH and freeze in liquid nitrogen or methanol dry ice exercise caution when handling liquid nitrogen it can cause severe burns if it comes in contact with the skin wear appropriate protective equipment Thaw the frozen lysate at 42 C Repeat this freeze thaw 2 3 additional times and pellet the insoluble protein in a refrigerated microcentrifuge for 10 minutes at maximum speed Remove the supernatant to a fresh labeled tube Resuspend the pellet in 100 pl of Laemmli Buffer To 100 ul of supern
20. rward primer RBS GGG GGT TCT Gly Gly Ser GGA CAG CAA Gly Gln Gln Xho I Sac I Bgl I ro 509 CCG AGC TCG AGA ro 557 CTG Leu Multiple Cloning Site of pTrcHis C 361 413 ATG Met GGT Gly 461 Ser Ser Arg AAAATTAAAG AGGTATATAT TA Mini cistron m Met ATG TAT CGA 1 Polyhistidine 6xHis region CAT His ATG Met TCT Ser CAT CAT CAT His His His CAT CAT His Xpress Epitope EK recognition sequence GGT ATG GCT AGC ATG ACT His Gly Met Ala Ser Met Thr Bam HI PTA AAT AAG GAG GAA TAA ACC yr Arg Leu Asn Lys Glu Glu I GGT CGG GAT Gly Arg Asp Pst I I GCA GCT GGT Ala Ala Gly CTG Leu Kpn I TAC GAC GAT n yr Asp Asp EcoR I l ACC ATA TGG GAA Thr Ile Trp Glu GAC GAT AAG Asp Asp Lys BstB I Hind II I I TTC GAA GCT Phe Glu Ala 1 GAT Asp EK cleavage site TGG Trp TTT Phe AAAATTAAAG AGGTATATAT TA GGG GGT Gly Gly GGA CAG Gly Gln BamH I TGG CGG Trp Arg ATG Met AGA GAA GAT Arg Glu Asp TTT CAG CCT GAT Phe Gly Pro Asp ACA GAT TAA Thr Asp ATC Below is the multiple cloning site for pTrcHis C Restriction sites are labeled to indicate the actual cleavage site Boxed nucleotides indicate the variable region Sequencing and functional testing have confirmed the multiple cloning site The complete sequence of pTrcHis C is available
21. s in 4 ml of ice cold 50 mM CaCl2 Keep the cells on ice Aliquot 100 pl of CaCl2 treated cells for each transformation into a pre chilled on ice Falcon 2059 tube or equivalent Add transforming DNA 10 100 ng in 1 10 ul and incubate on ice for 30 minutes After 30 minutes on ice heat shock the cells at 42 C for 45 seconds in a water bath After the heat shock return the tube s to ice for an additional 2 minutes Add 1 ml of SOC media and incubate the cultures for 1 hour at 37 C with vigorous shaking gt 200 rpm in a rotary shaking incubator Plate appropriate amounts of cells onto SOB or LB plates containing ampicillin 50 pg ml Grow 1 to 2 ml of the strain to be frozen in rich bacterial media e g SOB see page 9 for recipe overnight with antibiotic selection when appropriate Combine 0 85 ml of the overnight culture with 0 15 ml of sterile glycerol sterilized by autoclaving Mix the culture well by vortexing Transfer to an appropriate freezing vial Freeze in an ethanol dry ice bath or liquid nitrogen and then transfer to 80 C for long term storage Bacterial Alkaline Lysis Miniprep Procedure Grow 2 ml of bacterial culture LB broth with the appropriate antibiotic at 37 C overnight in a rotary shaking incubator Decant 1 5 ml of the culture into a microcentrifuge tube and spin it for 10 seconds Discard the supernatant leaving 50 100 ul of medium in the tube Vortex the tube to completely re
22. suspend the cells Add 300 ul of TENS solution 10 mM Tris HCl pH 7 5 1 mM EDTA 0 1 N NaOH 0 5 SDS then vortex the tube for 2 5 seconds or until the mixture becomes viscous Add 150 ul of 3 M sodium acetate pH 5 2 then vortex the tube for 2 5 seconds to mix completely Centrifuge the tube for 2 minutes in a microcentrifuge to pellet the cell debris and the chromosomal DNA Transfer the supernatant to a fresh microcentrifuge tube add 900 ul of cold 100 ethanol and mix well Freeze the solution on dry ice Centrifuge the tube for 5 minutes to pellet the plasmid DNA and the RNA The pellet should have a white appearance Discard the supernatant and rinse the pellet twice with 1 ml of 70 ethanol Remove the residual ethanol after another quick spin Resuspend the pellet for further analysis in 20 50 ul of TE buffer pH 8 0 10 mM Tris HCl pH 8 0 1 mM EDTA pH 8 0 or sterile water containing RNase A at a concentration of 100 ug ml Recipes SOB For 1 Liter SOC For 1 Liter LB For 1 Liter Ampicillin 50 mM CaCl 250 mM KCI To 950 ml of deionized water add 20 0 g Tryptone 5 0 g Yeast Extract 0 5 g NaCl 1 Mix the solution until dissolved 2 Add 10 ml of a 250 mM solution of KCl 3 Adjust the pH to 7 0 with 5 N NaOH approximately 0 2 ml 4 If making solid media for plates or top agar add 15 g of agar after adjusting the pH e Adjust the volume to 1000 ml and sterilize by autoclavin
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