pTrcHis2 - Thermo Fisher Scientific
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1. 4 You should be able to determine the optimal time point for maximum expression Use the conditions determined above to grow and induce 50 mL of cells This is the largest culture volume to use with the 2 mL prepacked columns included in the ProBond Purification System If you need to purify larger amounts of recombinant protein you may need more ProBond resin See page 11 for ordering information 1 Inoculate 2 mL of SOB or LB containing 50 pg mL ampicillin with a single recombinant E coli colony Grow overnight at 37 C with shaking 225 250 rpm The next day inoculate 50 mL of SOB or LB containing 50 pg mL ampicillin with 1 mL of the overnight culture 4 Grow the culture at 37 C with vigorous shaking to an OD600 0 6 the cells should be in mid log phase 5 Add IPTG to a final concentration of 1 mM 0 5 mL of a 100 mM IPTG stock to 50 mL 6 Grow at 37 C with shaking until the optimal time point is reached Harvest the cells by centrifugation 3 000 x g for 10 minutes at 4 C TM 7 Atthis point you may proceed directly to purification ProBond Purification System manual or store at 80 C for future use Expression of your recombinant protein can be detected using an antibody to the myc epitope encoded in the C terminal fusion peptide In addition the metal binding domain allows simple one step purification of your recombinant protein by Immobilized Metal Affinity Chromatography IMAC using2. invitrogen Corporate Headquarters 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
3. binding domain SE eee CAT CAT CAT CAT CAT TGA GTTTA His His His His His Multiple Cloning Below is the multiple cloning site for pTrcHis2 B Restriction sites are labeled to Site of pTrcHis2 B indicate cleavage site The boxed sequence is the variable region that facilitates in 361 413 469 518 frame cloning with the C terminal peptide This variable region is located between the Hind III site and the myc epitope The multiple cloning site has been confirmed by sequencing and functional testing TrcHis forward priming site Mini cistron Neo I RBS RBS AAAATTAAAG AGGTATATAT TA ATG TAT CGA TTA AAT AAG GAG GAA TAA ACC Met Tyr Arg Leu Asn Lys Glu Glu Samii I Xho I Ban I Bgl II el Asp718 I Kpn I EcoR I a I Hind HI 200 I ATG GATCCGAGCT CGAGATCTGC NGCTEETACT ATATGGGAAT TCGAAGCTTT CTA Met myc epitope tag Sal I I 1 GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT AGC GCC GTC GAC TAT Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His ProBond binding domain 1 CAT CAT CAT CAT CAT TGA GTTTA His His His His His Continued on next page Cloning into pTrcHis2 Continued Multiple Cloning Below is the multiple cloning site for pIrcHis2 C Restriction sites are labeled to Site of pTrcHis2 C indicate cleavage site The boxed sequence is the variable region that facilitates in 361 413 461 510 frame cloning with the C terminal peptide This variable region is
4. SOB Medium with Ampicillin 10 LB Medium per liter 1 Tryptone 0 5 Yeast Extract 0 5 NaCl pH 7 0 1 For 1 liter dissolve 10 g tryptone 5 g yeast extract and 5 g NaCl in 950 mL deionized water 2 Adjust the pH of the solution to 7 5 with 5 M NaOH and bring the volume to 1 liter 3 Autoclave for 20 minutes on liquid cycle 4 Let solution cool to 55 C Add ampicillin to a final concentration of 50 pg mL 5 Store the medium at 4 C Medium is stable for only 1 2 weeks LB Medium per liter 1 Tryptone 0 5 Yeast Extract 0 5 NaCl 1 5 Agar pH 7 0 1 For 1 liter dissolve 10 g tryptone 5 g yeast extract and 5 g NaCl in 950 mL deionized water 2 Adjust the pH of the solution to 7 5 with 5 M NaOH add 15 g agar and bring the volume to 1 liter 3 Autoclave for 20 minutes on liquid cycle Let agar cool to 55 C Add ampicillin to a final concentration of 50 ug mL 5 Pour into 10 cm petri plates Let the plates harden then invert and store at 4 C Plates containing ampicillin are stable for 1 2 weeks SOB per liter 2 Tryptone 0 5 Yeast Extract 0 05 NaCl 2 5 mM KCI 10 mM MgCl 1 Dissolve 20 g tryptone 5 g yeast extract and 0 5 g NaCl in 950 mL deionized water 2 Make a 250 mM KCI solution by dissolving 1 86 g of KCI in 100 mL of deionized water Add 10 mL of this stock KCI solution to the solution in Step 1 3 Adjust pH to 7 5 with 5 M NaOH and add deionized water
5. Invitrogen s ProBond Resin for ordering see page 11 Appendix pTrcHis2 Vectors Map of pTrcHis2 The figure below summarizes the features of the pTrcHis2 vectors The sequences for all three pTrcHis2 vectors can be downloaded from our website www invitrogen com or by contacting Technical Support see page 12 Details of each multiple cloning site are shown on pages 3 4 8 Prowl lac OM anti Y 910 RBST aTe MCS myc 6xHis term pTrcHis2 A B C 4 4 kb Comments for pTrcHis2 A 4406 nucleotides Xba is only found trc promotor region bases 190 382 in pTrcHis2 B 35 region bases 193 198 10 region bases 216 221 t SnaB is only found lac operator lacO bases 228 248 in pTrcHis2 C rrnB antitermination signal bases 264 333 gene 10 region bases 346 354 Ribosome binding site bases 369 373 pTrcHis forward priming site bases 370 390 Minicistron ORF bases 383 409 Reinitiation RBS bases 398 403 Expression ATG bases 413 415 Multiple cloning site bases 411 464 myc epitope bases 471 503 Polyhistidine tag bases 516 533 mycHis reverse priming site bases 508 527 rrnB T1 and T2 transcriptional terminators bases 639 796 Ampicillin resistance ORF bases 1076 1936 pBR322 origin bases 2081 2754 Lac Repressor lacl9 ORF bases 3408 4367 Continued on next page pTrcHis2 Vectors Continued Features of pTrcHis2 The important elements of pTrcHis2 A 4406 bp pTrcHis2 B 4404 bp and pTrcHi
6. of cells centrifuge at maximum speed in a microcentrifuge for 30 seconds and aspirate the supernatant Freeze the cell pellet at 20 C This is the zero time point sample Add IPTG to a final concentration of 1 mM 0 1 mL of a 100 mM IPTG stock to 10 mL and grow at 37 C with shaking 8 Take 1 mL samples every hour for 5 hours or more and treat as in Steps 5 and 6 Label each tube to correspond to the number of hours postinduction 1 When all the time points have been collected resuspend each pellet in 100 pL of 1X SDS PAGE sample buffer Boil 5 minutes and centrifuge briefly Analyze 5 uL of each sample on an appropriate SDS PAGE gel Continued on next page Expression Continued Analysis of Time Point Samples Expression of Recombinant Protein Detection and Purification of Recombinant Proteins 1 Stain the gel with Coomassie blue and look for a band of increasing intensity in the expected size range for the recombinant protein Note The myc epitope and polyhistidine region contribute 2 5 kDa to your protein Be sure and account for any additional amino acids at the N terminus and between the 3 cloning site and the myc epitope 2 Use the negative control to distinguish recombinant proteins from background proteins 3 Use the positive control to confirm that growth and induction was done properly The positive control should yield a 120 kDa protein with maximum expression occurring between 3 4 hours
7. to 1 liter 4 Autoclave this solution cool to 55 C and add 10 mL of sterile 1 M MgCI2 You may also add ampicillin to 50 ug mL 5 Store at 4 C Medium is stable for only 1 2 weeks Accessory Products Introduction Primers Antibodies for Detection The following products may be used with the pTrcHis2 vectors For details visit www invitrogen com or contact Technical Support page 12 Product Quantity Catalog no ProBond Purification System 6 purifications K850 01 50 mL R801 01 ProBond Resin 150 mL R801 15 Purification Columns 50 R640 50 One Shot Top 10 Electrocomp Cells 10 x 50 pL C4040 50 One Shot Top 10 Chemically Competent Cells 10 x 50 pL C4040 10 For your convenience Invitrogen offers a custom primer synthesis service Visit www invitrogen com for more details Invitrogen offers the Anti myc or Anti His C term antibodies to detect your recombinant fusion protein Horseradish peroxidase HRP and alkaline phosphatase AP conjugated antibodies are available for convenient one step detection Antibody Epitope Catalog no Anti myc Detects a 10 amino acid epitope R950 25 Anti myc HRP derived from c myc Evan et al 1985 R951 25 Anti myc AP EOE R952 25 Anti His C term Detects the C terminal polyhistidine R930 25 Anti His C term HRP t38 requires the free carboxyl group R931 25 for detection Lindner et al 1997 Anti
8. 322 derived expression vectors designed for efficient recombinant protein expression and purification in E coli High levels of expression are possible using the trc 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 To regulate expression the gene encoding Lac repressor lacl4 is provided in the pTrcHis2 vectors allowing regulation of the trc promoter regardless of whether the host strain contains a gene encoding the Lac repressor Isopropyl B D thiogalactopyranoside IPTG is used to induce expression of your gene Translation is enhanced by the bacteriophage T7 gene 10 translation enhancer and a minicistron that provides highly efficient translational restart into the open reading frame of the multiple cloning site DNA inserts are positioned downstream and in frame with the initiation ATG and a C terminal fusion peptide The C terminal peptide encodes the myc epitope and six histidine residues that function as a metal binding site in the expressed protein Methods Cloning into pTrcHis2 General Molecular Biology Techniques Maintaining pTrcHis2 E coli Strain Cloning in the pTrcHis2 Vectors For help with DNA ligations E coli transformations restriction enzyme analysis DNA sequencing and DNA biochemist
9. His C term AP HHHHHH COOH R932 25 11 Technical Support Web Resources Visit the Invitrogen website 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 or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters 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 supportQinvitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Certificate of Analysis Limited Warranty 12 Material Safety Data Sheets MSDSs are available on our website at www invitrogen com msds The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to w
10. Specific for myc Proto oncogene Product Mol Cell Biol 5 3610 3616 Li S C Squires C L and Squires C 1984 Antitermination of E colirRNA Transcription is Caused by a Control Region Segment Containing Lambda nut like Sequences Cell 38 851 860 Lindner P Bauer K Krebber A Nieba L Kremmer E Krebber C Honegger A Klinger B Mocikat R and Pluckthun A 1997 Specific Detection of His tagged Proteins With Recombinant Anti His Tag scFv Phosphatase or scFv Phage Fusions BioTechniques 22 140 149 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 Edition Plainview New York Cold Spring Harbor Laboratory Press 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 2009 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 14
11. er only in the spacing between the sequences that code for the multiple cloning site and the C terminal peptide For proper expression first determine which restriction sites are appropriate for ligation and then which vector will preserve the reading frame at BOTH the 5 and the 3 ends You may have to use PCR to create a fragment with the appropriate restriction sites to clone in frame at both ends Be sure that there is no stop codon in the open reading frame of your gene Continued on next page Cloning into pTrcHis2 Continued Multiple Cloning Below is the multiple cloning site for pTrcHis2 A Restriction sites are labeled to Site of pTrcHis2 A indicate cleavage site The boxed sequence is the variable region that facilitates in 361 413 461 510 frame cloning with the C terminal peptide This variable region is located between the Hind III site and the myc epitope The multiple cloning site has been confirmed by sequencing and functional testing pTrcHis forward priming site Mini cistron RBS AAA A S Neg I AAAATTAAAG AGGTATATAT TA ATG TAT CGA TTA AAT AAG GAG GAA TAA ACC Met Tyr Arg Leu Asn Lys Glu Glu BamH I Xho I Sac I Bel II Pl Asp718 I Kpn I EcoRI Bs I Hind Ill ATG GATCCGAGCT CGAGATCTGC AGCTGGTACC ATATGGGAAT TCGAAGCIT G_GGCCC Met myc epitope tag Sal I I GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT AGC GCC GTC GAC CAT Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His ProBond
12. es and overproduces the Lac repressor protein pTrcHis2 lacZ Description Map of Control Vector Comments for pTrcHis2 lacZ pIrcHis2 lacZ is a 7552 bp control vector containing the gene for B galactosidase It was constructed by digesting pTrcHis2 A with BamH I and Xho I A 3 2 kb BamH I Xho I fragment containing the lacZ gene was then ligated into pTrcHis2 A The vector expresses a 120 kDa protein The figure below summarizes the features of the pTrcHis2 lacZ vector The nucleotide sequence for pTrcHis2 lacZ may be downloaded from our website www invitrogen com or by contacting Technical Support see page 12 Nco BamH Xho Sac Bg II Hind III Piro gm lac O An g10 RBSIRWMMEE ATG MCS myc 6xHis AEO pTrcHis2 lacZ 7 6 kb 7552 nucleotides trc promotor region bases 190 382 Expression ATG bases 413 415 35 region bases 193 198 lacZ ORF bases 467 3523 10 region bases 216 221 myc epitope bases 3617 3649 lac operator lacO bases 228 248 Polyhistidine tag bases 3662 3679 rrnB antitermination region bases 264 333 mycHis reverse priming site bases 3654 3673 gene 10 region bases 346 354 Ampicillin resistance ORF bases 4222 5082 Ribosome binding site bases 369 373 pBR322 origin bases 5227 5900 Minicistron ORF bases 383 409 Lac Repressor lacl4 ORF bases 6554 7513 Reinitiation RBS bases 398 403 Recipes LB Medium with Ampicillin LB Agar Plates with Ampicillin
13. invitrogen pTrcHis2 A B and C Catalog no V365 20 Rev date 26 August 2009 Manual part no 25 0096 MAN0000022 ii Table of Contents Kit Contents and Storage svn rsca een atmen nun apa unable iv INTFOCUCTION es hewasuwennansneh napibnchbuseanstasehasenusohusenmmennsenisennsenineninas 1 Product Overview NT 1 MICE OOS needed kaken dn EEE LIE 2 Cl ning into TCH IS ots seen ana aan alarm nalen beruhen 2 Expression sses emendat navenante ident hots bent te neten ag 5 poo lol EE ino NN ME 7 SAB Kel m TEZAVE r AEE E skam ssladinadekenne 7 pIrcHisZ Mach iii a In En weeken verte 9 RRA 10 Accessory Products A ON 11 Technical Support sassen delende ineen dead denn IRRE ea 12 P rchaser Notifica 2 sns tad 13 References 22a ebene reisen 14 iii Kit Contents and Storage Shipping and Storage Kit Contents pTrcHis2 vectors are shipped on wet ice Upon receipt store vectors at 20 C All vectors are supplied as detailed below Store the vectors at 20 C Note For long term storage of your stab we recommend preparing a glycerol stock immediately upon receipt and storing at 80 C Vector Composition Amount 40 uL of 0 5 ug uL vector in 10 mM Tris pTrcHis2 A B and C HCI 1 mM EDTA pH 8 0 20 ug 40 uL of 0 5 ug uL vector in 10 mM Tris Pt HCL 1 mM EDTA pH 8 0 20 ug TOP10 E coli stab 1 stab Introduction Product Overview pTrcHis2 The pTrcHis2 plasmids are pBR
14. located between the Hind III site and the myc epitope The multiple cloning site has been confirmed by sequencing and functional testing TrcHis forward priming site sed p p 5 Mini cistron AAAATTAAAG AGGTATATAT TA ATG TAT CGA TTA AAT AAG GAG GAA TAA ACC Met Tyr Arg Leu Asn Lys Glu Glu BamH I Xho I age I Bel II Psl Asp718 I Kpn I EcoR I ae I Hind Il SnaBl 4 ATG GATCCGAGCT CGAGATCTGC NGCTEGTACT ATATGGGAAT TCGAAGCTITA CGTA Met myc epitope tag Sal I l m Nl men GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT AGC GCC GTC GAC CAT Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His ProBond binding domain TE BE CAT CAT CAT CAT CAT TGA GTTTA His His His His His E coli Transform your ligation mixtures into a competent recA endA E coli strain e g Transformation TOP10 DH5a and select on LB plates containing 50 100 ug mL ampicillin Select 10 20 clones and analyze for the presence and orientation of your insert We recommend that you sequence your construct with the pTrcHis Forward ar primer to confirm that your gene is in frame with the initiation ATG and the Tal E C terminal peptide For ordering primers see page 11 Preparing a Once you have obtained your construct we recommend that you store your clone Glycerol Stock as a glycerol stock 1 Grow 1 to 2 mL of the strain containing your construct in pTrcHis2 to saturation Combine 0 85 mL of the stationary cu
15. lture with 0 15 mL of sterile glycerol Mix the solution by vortexing Transfer to an appropriate vial for freezing and cap Or oe DD Freeze in an ethanol dry ice bath or liquid nitrogen and then transfer to 80 C for long term storage Expression Introduction Materials Needed Pilot Expression Preparing Time Point Samples Since each recombinant protein has different characteristics that may affect optimum expression it is helpful to run a time course of expression to determine the optimal time for maximum expression of your particular protein A mock expression consisting of the pTrcHis2 vector alone should be done as a negative control pTrcHis2 lacZ is provided for use as a positive expression control see page 9 Transform all plasmids into TOP10 E coli or similar strains to analyze expression see page 11 e SOB or LB containing 50 ug mL ampicillin see Recipes page 10 e 37 C shaking incubator e 100 mM IPTG e 1X and 2X SDS PAGE sample buffer e Reagents and apparatus for SDS PAGE gel 1 For each strain inoculate 2 mL of SOB or LB containing 50 pg mL ampicillin with a single recombinant E coli colony Grow overnight at 37 C with shaking 225 250 rpm The next day inoculate 10 mL of SOB or LB containing 50 pg mL ampicillin with 0 2 mL of the overnight culture 4 Grow the culture at 37 C with vigorous shaking to an OD600 0 6 the cells should be in mid log phase 5 Remove a 1 mL aliquot
16. resentatives Life Technologies Corporation shall have 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 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 13 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 New York Greene Publishing Associates and Wiley Interscience 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 Evan G I Lewis G K Ramsay G and Bishop V M 1985 Isolation of Monoclonal Antibodies
17. ry see Molecular Cloning A Laboratory Manual Sambrook et al 1989 or Current Protocols in Molecular Biology Ausubel et al 1994 Use the supplied 0 5 ug uL stock solution in TE pH 8 0 to transform a recA end A E coli strain like TOP10 DH5a or equivalent Transformants are selected on LB plates containing 50 100 pg mL ampicillin TOP10 is provided for growth and maintenance of these plasmids This strain is provided as a convenience for those who do not have access to other E coli strains Many E coli strains are suitable for the growth of this vector We recommend that you propagate vectors containing inserts in recombination deficient recA endonuclease A deficient end A E coli strains Genotype EmcrA A mrr hsdRMS mcrBC 80lacZAM15 AlacX74 recA1 araD139 A ara leu 7697 galU galK rpsL endA1 nupG 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 For your convenience TOP10 is available as competent cells from Invitrogen see page 12 To generate recombinant proteins that are expressed correctly and contain the C terminal fusion peptide it is necessary to clone in frame with BOTH the initiation ATG bp 413415 and the C terminal peptide To facilitate cloning the pTrcHis2 vector is provided in three different reading frames They diff
18. s2 C 4405 bp are described in the following table 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 lacO Permits binding of the Lac repressor to repress transcription rrnB antitermination region 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 Initiation ATG Provides a translation initiation site for the fusion protein Multiple cloning site Allows insertion of your gene for expression C terminal myc epitope tag Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Allows detection of the fusion protein by the Anti myc Antibody Evan et al 1985 for ordering see page 11 C terminal polyhistidine region Formation of the metal binding site for affinity purification of recombinant protein Ampicillin resistance gene B lactamase Allows selection of the plasmid in E coli pBR322 origin Low copy replication and growth in E coli lacld gene Encod
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