GeneArt® TAL User Manual - Thermo Fisher Scientific
Contents
1. Plasmid DNA for transfection in eukaryotic cells must be very clean and free from contamination with phenol and sodium chloride Contaminants will kill the cells and salt may interfere with lipid complexing decreasing transfection efficiency We recommend isolating plasmid DNA using the S N A P MidiPrep Kit Cat no K1910 01 PureLink HQ Mini Plasmid Purification Kit Cat no K2100 01 or CsCl gradient centrifugation If you used the pENTR gus control vector in an LR recombination reaction with a destination vector you can use the resultant expression clone as a positive control for mammalian cell transfection and expression A successful transfection will result in B glucuronidase expression that can be detected by western blot or functional assay We recommend using Lipofectamine 3000 Reagent Catalog no L3000015 or the transfection method recommended by the supplier of the cell type being used For more information refer to www lifetechnologies com transfection or contact Technical Support see page 28 GeneArt PerfectMatch TALs and GeneArt Precision TALs 15 Appendix Map of N TAL Fokl Entry Vector N TAL Fokl Entry The map below shows the elements of the N TAL FokI Entry Vector The region Vector map of the entry clone corresponding to the TAL is variable depending upon the length of the sequence you ordered The complete sequence for your clone in gb format is available on the disk provided with your clon2. directed towards the submission of data to the United States Department of Agriculture or any equivalent regulatory agency outside of the United States in support of an application for clearance approval or deregulation by such agency d screening or profiling of more than 10 000 distinct compounds high throughput screening and or e scale up activities the primary focus of which is to increase from small scale to production scale Plant means any eukaryotic plants plant tissues or plant cells including green algae belonging to the plant kingdom defined as Viridiplantae including any progeny propagated through any number of generations or unmodified derivatives of such plants tissues or cells For clarity fungi or blue green algae are specifically excluded from this definition For information on obtaining additional rights to TAL Effector technology for any use not permitted herein except use in Plants please contact Life Technologies at outlicensingldlifetech com For information on obtaining additional rights to TAL Effector technology for any use in Plants not permitted herein please contact Two Blades Foundation at infold2blades org or Two Blades Foundation 1630 Chicago Avenue Suite 1907 Evanston IL 60201 USA For information on donating mice to The Jackson Laboratory please visit their website at http www jax org grc index html Limited Use Label License No 521 GeneArt Precision TALEN Products The pur
3. following Gateway adapted entry vectors TAL Fokl TAL VP16 TAL VP64 TAL KRAB and TAL MCS GeneArt Precision TALs engineered with the FokI nuclease can be used for targeting specific genes for silencing Fokl is a type IIS restriction endonuclease from Flavobacterium okeanokoites consisting of an N terminal DNA binding domain and a non specific DNA cleavage domain at the C terminal A FokI nuclease pair binds to duplex DNA at the target sites designated by the DNA binding domains to cleave the DNA Functional DNA binding domain domain GeneArt Precision TALs engineered with the VP16 or VP64 activators can be used to increase the expression level of endogenous or recombinant genes VP16 is a trans acting protein originating from the herpes simplex virus that forms a complex with host transcription factors to induce immediate early gene transcription VP64 is a tetrameric form of the VP16 minimal activation domain Functional DNA binding domain domain GeneArt Precision TALs engineered with the KRAB repressor can be used to down regulate the expression level of endogenous or recombinant genes Functional DNA binding domain domain GeneArt PerfectMatch TALs and GeneArt Precision TALs TAL MCS The Gateway Technology LR recombination reaction GeneArt Precision TALs that include a multiple cloning site MCS allow the user to clone any desired effector domain and target the protein to any locus withi
4. of the vector select for transformants in media containing 50 100 pg mL ampicillin and 15 30 pg mL chloramphenicol IMPORTANT Do NOT use general E coli cloning strains including TOP10 or DH5a for propagation and maintenance of destination vectors as these strains are sensitive to the toxic effects of the ccdB gene GeneArt PerfectMatch TALs and GeneArt Precision TALs 9 Create an expression clone continued Gateway The LR reaction facilitates recombination of an attL substrate entry clone with an attR substrate destination vector to create an attB containing expression recombination clone see diagram below This reaction is catalyzed by LR Clonase II Enzyme reactions A Mix aftL aftL attR attR attB attB attP attP LR Clonase II destination al expression by product vector clone Recombination In the following example the recombination region of the expression clone resulting from the LR reaction between a TAL entry clone and the Gateway region of the 5 a pcDNA DEST40 destination vector sequence is shown expression clone Features of the recombination region e Shaded regions correspond to those DNA sequences transferred from the entry clone into the destination vector by recombination e Non shaded regions are derived from the destination vector e The underlined nucleotides flanking the shaded region correspond to bases 918 and 2601 respectively of the Gateway pcDNA DEST40 destination vect
5. patch plate to preserve the colonies for further analysis Incubate reaction for 10 minutes at 94 C to lyse cells and inactivate nucleases Amplify for 20 to 30 cycles For the final extension incubate at 72 C for 10 minutes Store at 4 C Dye OM ES 209 Visualize by agarose gel electrophoresis The ccdB gene mutates at a very low frequency resulting in a very low number of false positives True expression clones will be ampicillin resistant and chloramphenicol sensitive Transformants containing a plasmid with a mutated ccdB gene will be ampicillin and chloramphenicol resistant To check your putative expression clone test for growth on LB plates containing 30 ng mL chloramphenicol A true expression clone will not grow in the presence of chloramphenicol GeneArt PerfectMatch TALs and GeneArt Precision TALs Transfection Introduction Plasmid preparation Positive control Methods of transfection Once you have generated your expression clone you are ready to transfect the plasmid into the mammalian cell line of choice You may perform transient transfection experiments or use Geneticin selection to generate stable cell lines The neomycin resistance gene in pcDNA dest 40 Gateway vector allows for the selection of stable cell lines using Geneticin antibiotic We recommend that you include a positive control see below and a negative control mock transfection in your experiment to evaluate your results
6. recombination reaction using LR Clonase Enzyme Mix if desired To use LR Clonase Enzyme Mix follow the protocol provided with the product Do not use the protocol for LR Clonase II Enzyme Mix provided in this manual as reaction conditions differ You should have the following materials on hand before beginning e Purified plasmid DNA of your entry clone 50 150 ng uL in TE pH 8 0 e Destination vector 150 ng L in TE pH 8 0 e LR Clonase II Enzyme Mix Cat no 11791 020 11791 100 e TE Buffer pH 8 0 10 mM Tris HCI pH 8 0 1 mM EDTA e Proteinase K solution supplied with the LR Clonase II Enzyme Mix e pENTR gus positive control supplied with the LR Clonase II Enzyme Mix GeneArt PerfectMatch TALs and GeneArt Precision TALs 11 Perform the LR recombination reaction continued Set up the LR recombination reaction 12 Follow this procedure to perform the LR reaction between your entry clone and the destination vector If you want to include a negative control set up a separate reaction but omit the LR Clonase II Enzyme Mix Add the following components to 1 5 mL microcentrifuge tubes at room temperature and mix Set up an additional set of reactions for your negative control You will not add LR Clonase II Enzyme Mix to these reactions Component Sample Positive control Entry clone 50 150 ng reaction 1 7 uL Destination vector 150 ng L Tul Tul pENTR gus 50 ng uL 2 uL TE
7. 0 Antigen of Schistosoma japonicum Recognized by Resistant WEHI 129 J Mice is a Parasite Glutathione S transferase Proc Natl Acad Sci USA 83 8703 8707 Southern J A Young D F Heaney F Baumgartner W and Randall R E 1991 Identification of an Epitope on the P and V Proteins of Simian Virus 5 That Distinguishes Between Two Isolates with Different Biological Characteristics J Gen Virol 72 1551 1557 GeneArt PerfectMatch TALs and GeneArt Precision TALs 29 30 GeneArt PerfectMatch TALs and GeneArt Precision TALs GeneArt PerfectMatch TALs and GeneArt Precision TALs 31 For support visit lifetechnologies com support or email techsupport dlifetech com lifetechnologies com 04 September 2014 technologies
8. 6 Lamb BM Mercer AC Barbas CF 2013 3rd Directed evolution of the TALE N terminal domain for recognition of all 5 bases Nucleic Acids Res 41 21 9779 9785 Landy A 1989 Dynamic Structural and Regulatory Aspects of Lambda Site specific Recombination Ann Rev Biochem 58 913 949 Li T Huang S Zhao X Wright D A Carpenter S Spalding M H Weeks D P and Yang B 2011 Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes Nucleic Acids Res 39 6315 6325 Mak AN Bradley P Cernadas RA Bogdanove AJ Stoddard BL 2012 The crystal structure of TAL effector PthXo1 bound to its DNA target Science 335 716 719 Mussolino C and Cathomen T 2012 TALE nucleases tailored genome engineering made easy Curr Opin Biotechnol 23 5 644 650 Moscou M J and Bogdanove A J 2009 A simple cipher governs DNA recognition by TAL effectors Science 326 1501 Orosz A Boros I and Venetianer P 1991 Analysis of the Complex Transcription Termination Region of the Escherichia coli rrnB Gene Eur J Biochem 201 653 659 Ptashne M 1992 A Genetic Switch Phage Lambda and Higher Organisms Cambridge MA Cell Press Scholze H and Boch J 2011 TAL effectors are remote controls for gene activation Curr Opin Microbiol 14 47 53 Smith D B Davern K M Board P G Tiu W U Garcia E G and Mitchell G F 1986 Mr 26 00
9. Buffer pH 8 0 to8 uL 5 uL Remove the LR Clonase II Enzyme Mix from 20 C and thaw on ice 2 minutes Vortex the LR Clonase II Enzyme Mix briefly twice 2 seconds each time Add 2 uL of LR Clonase II Enzyme Mix to each sample or positive control reaction listed above Mix well by pipetting up and down Do not add LR Clonase II Enzyme Mix to negative control reactions Reminder Return LR Clonase II Enzyme Mix to 20 C immediately after use Incubate reactions at 25 C for 1 hour Note For most applications 1 hour will yield a sufficient number of colonies for analysis Depending on your needs the length of the recombination reaction can be extended up to 18 hours For large plasmids 210 kb longer incubation times will yield more colonies Add 1 uL of Proteinase K solution to each reaction Incubate for 10 minutes at 37 C Proceed to Transform competent E coli cells next page Note You may store the LR reaction at 20 C for up to 1 week before transformation if desired GeneArt PerfectMatch TALs and GeneArt Precision TALs Transform competent coli cells Introduction Once you have performed the LR recombination reaction transform chemically competent E coli with the resulting expression clone Materials needed You should have the following materials on hand before beginning Transformation 1 procedure 2s 3 4 5 6 7 8 9 LR recombination reaction from Step 7 previous pa
10. GGA TCC GAG CTC AAG Val Asp Gly Thr Glu Phe Leu Ile Asp Ser Thr Leu Glu Gly Ser Glu Leu ATC TAG CTA AGT AGA CCC AGC TTT CTT GTA CAA AGT TGG CAT TAT AAG Lys The multiple cloning site for the Truncated TAL MCS entry clone is shown below The sequence of the TAL C terminus is in bold The MCS is underlined Restriction sites are labeled to indicate the cleavage site TTT TTT CAG TGT CAC TCT CAC CCT GCC CAG GCC TTT GAT GAT GCC ATG ACA Phe Phe Gln Cys His Ser His Pro Ala Gln Ala Phe Asp Asp Ala Met Thr CAG TTT GGC ATG AGC AGA CAC GGA CTG CTG CAG CTG TTT AGA AGA GTG GGA Gln Phe Gly Met Ser Arg His Gly Leu Leu Gln Leu Phe Arg Arg Val Gly GTG ACA GAA CTG GAG GCC AGA TCC GGA ACC CTG CCT CCT GCC TCT CAG AGA Val Thr Glu Leu Glu Ala Arg Ser Gly Thr Leu Pro Pro Ala Ser Gln Arg Pmel HindIII Sail KpnI l l l TGG GAT AGG ATT CTG CAG GGT TCC CGT TTA AAC AAG CTT GTC GAC GGT ACC Trp Asp Arg Ile Leu Gln Gly Ser Arg Leu Asn Lys Leu Val Asp Gly Thr SacI EcoRI Clal Scal XhoI BamHI BglII l l l l l GAA TTC ATC GAT AGT ACT CTC GAG GGA TCC GAG CTC AAG ATC TTA GCT AAG Glu Phe Leu Ile Asp Ser Thr Leu Glu Gly Ser Glu Leu Lys TAG ACC CAG CTT TCT TGT ACA AAG TTG GCA TTA TAA GAA GeneArt PerfectMatch TALs and GeneArt Precision TALs Features of GeneArt Precision TALs entry vectors Common TAL entry The following elements are found in the GeneArt Precision TALs entry vectors vector features TAL Fokl TAL vp16 vp64 TAL MCS All features have
11. USER GUIDE GeneArt PerfectMatch TALs and GeneArt Precision TALs TAL effector expression system for genome editing Catalog Numbers 816508DE 816509DE 816510DE 816511DE 816512DE 816514DE 816516DE 816517DE 816518DE 816519DE 816010DE 816011DE Publication Number MANO0006670 Revision A 0 For Research Use Only Not for use in diagnostic procedures technologies Information in this document is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF Limited Use Label License No 406 GeneArt Precision TAL Effector Products The purchase of this product conveys to the buyer limited non transferable rights under certain TAL Effector technology owned by inventors from Martin Luther Universitat Halle Wittenberg and licensed to Life Technologies Corporation to use this product and components of this product only to perform internal research for the sole be
12. able depending upon the length of the sequence you ordered The complete sequence for your clone in gb format is available on the disk provided with your clone TAL KRAB Entry Vector E O O E C GeneArt PerfectMatch TALs and GeneArt Precision TALs Map of TAL MCS Entry Vector TAL MCS Entry The map below shows the elements of the TAL MCS Entry Vector The region of Vector map the entry clone corresponding to the TAL is variable depending upon the length of the sequence you ordered The complete sequence for your clone in gb format is available on the disk provided with your clone GeneArt PerfectMatch TALs and GeneArt Precision TALs 23 Multiple cloning site of TAL MCS Entry Vector Native TAL MCS 2849 2900 2951 3002 Truncated TAL MCS 1901 1952 2003 2054 2105 2156 24 The multiple cloning site for the Native TAL MCS entry clone is shown below The sequence of the TAL C terminus is in bold The MCS is underlined Restriction sites are labeled to indicate the cleavage site GAT CCT TTT GCC GGA ACA GCC GAT GAT TIC CCT GCC TTT AAT GAG GAA GAA Asp Pro Phe Ala Gly Thr Ala Asp Asp Phe Pro Ala Phe Asn Glu Glu Glu Pmel HindIII l l CTG GCC TGG CTG ATG GAA CTG CTG CCT CAG GGT TCC CGT TTA AAC AAG CTT Leu Ala Trp Leu Met Glu Leu Leu Pro Gln Gly Ser Arg Leu Asn Lys Leu EcoRI SacI Salil KpnI Clal Scal XhoI BamHI Bgl111 l l l l l l l GTC GAC GGT ACC GAA TTC ATC GAT AGT ACT CTC GAG
13. attL 1 bases 99 198 gus gene bases 228 2039 attL2 bases 2041 2140 pUC origin bases 2200 2873 C Kanamycin resistance gene bases 2990 3805 C C complementary strand GeneArt PerfectMatch TALs and GeneArt Precision TALs Accessory products Introduction Additional products Gateway destination vectors The products listed in this section may be used with GeneArt PerfectMatch TALs and GeneArt Precision TALs For more information refer to our Web site www lifetechnologies com or contact Technical Support see page 28 Many of the reagents suitable for use with the vectors are available separately Ordering information for these reagents is provided below For more information refer to our Web site www lifetechnologies com Item Quantity Catalog no Gateway LR Clonase Il Enzyme Mix 20 reactions 11791 020 100 reactions 11791 100 Library Efficiency DH5 a Competent Cells 5x 0 2 mL 18263 012 One Shot TOP10 Chemically Competent 20 reactions C4040 03 E coli One Shot TOP10 Electrocompetent F col 20 reactions C4040 52 One Shot MAX Efficiency DH10B T1 20 reactions 12331 013 Phage Resistant col Ampicillin 200 mg 11593 027 Lipofectamine 3000 Transfection Reagent 1 5 mL L3000015 Kanamycin Sulfate 5g 11815 024 25g 11815 032 Kanamycin Sulfate 100X liquid 100 mL 15160 054 Geneticin Selective Antibiotic 1g 11811 023 59 11811 031 20 mL 50
14. been functionally tested Feature Description rrnB T1 and T2 transcription Protects the cloned gene from expression by vector encoded terminators promoters thereby reducing possible toxicity Orosz et al 1991 T7 promoter priming site C Allows n vitro transcription in the sense orientation and sequencing through the insert M13 Forward 20 priming site Allows sequencing in the sense orientation M13 Reverse C priming site Allows sequencing in the antisense orientation attL1 and attL2 sites Allows recombinational cloning of the gene of interest from an entry clone Landy 1989 Kanamycin resistance gene Allows selection of the plasmid in coli V5 epitope Allows detection of the recombinant fusion protein by the Anti V5 Gly Lys Pro lle Pro Asn Pro antibodies Southern et al 1991 Leu Leu Gly Leu Asp Ser Thr pUC origin Allows high copy number replication and growth in coli DNA binding domain Allows targeting of the TAL effector to specific DNA sequences DNA repeat variable domain TAL N term N terminus domain of the TAL containing translocation and nuclear localization signal tag NLS Truncated versions of the vector contain the SV40 nuclear localization signal NLS while native vectors contain the two endogenous NLS of the TAL TAL C term C terminus domain of the TAL containing activation domain C complementary strand Specific TAL entry The following features a
15. chase of this product conveys to the buyer limited non transferable rights under certain TALEN technology owned and or controlled by Cellectis SA including patents owned by the University of Minnesota and lowa State University and licensed to Life Technologies Corporation to use this product and components of this product only to perform internal research for the sole benefit of the buyer The buyer may also use standard molecular biology techniques to make additional copies of this product for purposes of internal research for the sole benefit of the buyer except the buyer may not modify the sequence of the TAL effector within this TALEN product Any buyer that is a for profit entity may not use this product its components or materials or cells made using this product or its components to generate any animal without first obtaining additional rights from Life Technologies The buyer cannot sell or otherwise transfer a this product b its components or c materials cells or organisms made using this product or its components including but not limited to Plants in all cases hereunder to a third party or otherwise use this product its components or materials cells or organisms made using this product or its components for any Commercial Purpose or Development Purpose with the sole exception that buyer may transfer this product its components and or materials cells or organisms made using this product or its components to i the buyer
16. ciency is thought to differ since strong and weak binding motifs exist The A and T binding motifs are thought to fall within the weak binder category while the C and G binding motifs are thought to be strong binders Stretches of more than 5 weak binders should be avoided at the extreme 5 end of the binding domain not counting the 5 T or if they are not flanked by Cs It is recommended to select a TAL effector with a DNA binding domain composed of mixed binding motifs for best results In the context of the living cell DNA accessibility also determines TAL effector efficiency It is possible that chromatin DNA methylation and or proteins bound to the DNA may interfere with TAL binding Although promoter structure varies and specific rules regarding design are currently lacking it is recommended that TAL transcription factors used for transcriptional activation of natural promoters be positioned upstream of the TATA box or in some cases downstream of the transcriptional start site Selecting a target site directly over the TATA box or other known transcription factor binding site is not recommended Be sure that the natural ATG is present and that no premature ATG which may interfere with the natural translational start is transcribed GeneArt PerfectMatch TALs and GeneArt Precision TALs Create an expression clone Introduction Resuspend the vectors Propagate the vectors To create an expression
17. clone perform the LR recombination reaction to transfer the gene of interest from the Gateway adapted entry vector into your destination vector of choice We recommend that you review this section and the next section entitled Perform the LR recombination reaction pages 11 12 before proceeding Note This step is not required when using the GeneArt PerfectMatch TAL N TAL FokI CMV Each destination vector is supplied as 6 ug of lyophilized plasmid To use resuspend the destination plasmid in 40 pL of TE Buffer pH 8 0 to a final concentration of 150 ng pL Note Destination vectors are supplied as supercoiled plasmids The linearization of the destination vector is NOT required to obtain optimal results for any downstream application Entry clone Propagate and maintain your entry clone using a recA endA E coli strains like TOP10 TOP10F DH5a JM109 or equivalent for transformation Select transformants on LB plates containing 50 100 pg mL kanamycin Prepare a glycerol stock of each plasmid for long term storage Destination vector If you wish to propagate and maintain your destination vectors prior to recombination we recommend using One Shot ccdB Survival T1 Chemically Competent E coli Cat no C7510 03 for transformation The One Shot ccdB Survival T1 E coli strain is resistant to the toxic effects of the ccdB gene and can support the propagation of plasmids containing the ccdB gene To maintain the integrity
18. e N TAL Fok1 Entry Vector 4349 bp P f L term V5 16 GeneArt PerfectMatch TALs and GeneArt Precision TALS Map of N TAL Fokl CMV Expression Vector N TAL Fokl CMV The map below shows the elements of the N TAL FokI CMV Expression Vector The region of the entry clone corresponding to the TAL is variable depending upon the length of the sequence you ordered The complete sequence for your clone in gb format is available on the disk provided with your clone Expression Vector map expression vector 5239 bp N TAL Fok1 CMV term 5 i GeneArt PerfectMatch TALs and GeneArt Precision TALS 17 Features of GeneArt PerfectMatch TAL vectors Common N TAL Fokl Fokl CMV vector features The following elements are found in the GeneArt PerfectMatch TALs N TAL Fokl and N TAL FokI CMV Feature Description T7 promoter priming site Allows n vitrotranscription in the sense orientation and sequencing through the insert V5 epitope Gly Lys Pro lle Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Allows detection of the recombinant fusion protein by the Anti V5 antibodies Southern et al 1991 pUC origin Allows high copy number replication and growth in coli DNA binding domain Allows targeting of the TAL effector to specific DNA sequences DNA repeat variable domain TAL N term N terminus domain of the TAL containing translocation and nuclea
19. etech com For information on obtaining rights to TALEN technology for any use in the therapeutic field please contact Cellectis SA at business developmentldcellectis com For information on obtaining rights to TALEN technology for any use in Plants not permitted herein please contact Cellectis Plant Sciences Inc at businessdevelopment_cps dcellectis com 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified TALEN is a trademark of Cellectis Contents Contents and Sto Ei A ae a ae a A 1 Introducir a ae 2 Produetiniorma UOM sensie ennaa enn eaa SASKE RAE AA EENE NNA SEE KANAS a SA NEAS DERSE RA E n PEASKE DESEORI AEREAS 2 Methods iii E E E A E E T T 6 Experimental Outlet ted EA E 6 Create a TAL SCQUENCE ecccceecceeecceeeceeeeeecteneeceaeeesaeeecaneesaaeeceaeescaeeecaaesscececaeeseaeessacecseaeeseaeessaeessueeeseeeeseeeess 7 Create an eA rE SS OE lO a e Ea ar aaae aaa E ar e ra a aaa aaea eiae 9 Perform the LR recombination reactionaire aAa E EEA AA E AA E EER 11 Transtorm COM pee A Co el ic 13 Analyze transformants nin a a aae T a ae a a ae a a a Aa o ae e enaena aek 14 Trastero e 15 AD PON GIX aer A A A AE da A 16 Map of NTA E FOR E VEC O dl ort tudes bol el led 16 Map of N TAL Fokl CMV Expression Vector cccccccesceeeeeeeeseeeeeeeeeeeeeeeaeeeeaeeseaeeeseceseeeeeseaeeseaeeseieeeeeeeesseeess 17 Featu
20. f they are not flanked by Cs It is recommended to select a TAL effector with a DNA binding domain composed of mixed GeneArt PerfectMatch TALs and GeneArt Precision TALs 7 GeneArt Precision TALs binding site rules binding motifs for best results In the context of the living cell DNA accessibility also determines TAL effector efficiency Chromatin structure DNA methylation and or proteins bound to the DNA may interfere with TAL binding The following are guidelines and rules for generating the Precision TAL sequence The GeneArt Precision TALs offering allows the construction of TAL effector functional proteins directed to either 18 or 24 base DNA target sites Each target site must be preceded by a 5 T because the N terminus of the TAL effector protein contains a conserved T binding motif The 5 T does not count as one of the 18 or 24 bases to be selected for targeting your specific site Nuclease pairs need to be designed with a spacing of 13 18 bp between the target sites on opposite strands of the DNA Both target sites must be preceded by a5 T The target sites can be either 18 or 24 bp in length The following image should be used as a reference for the orientation of the binding domains Functional DNA binding domain domain TNNNNNNNNNNNNNNNNNN oe 13 18 bp DOUDDOOGODOD INNNNNNNNNNNNNNNNNT The contribution of individual binding motifs within the DNA binding domain to TAL effector binding effi
21. fectMatch TALs provided in this vector can be directly used for expression in mammalian systems without the need for any intermediate sub cloning steps Optional PerfectMatch TAL cassette can be transferred directly into your expression vector of choice with the restriction enzymes Not I and Hind III Each target site sequence is preceded by a 5 N PerfectMatch TAL protein allows binding to a DNA sequence preceded by any DNA base The letter N represents any base of A G C or T The 5 N does not count as one of the 18 or 24 bases to be selected for targeting your specific site Design nuclease pairs with a spacing of 13 18 bp between the target sites on opposite strands of the DNA However we recommend a spacing of 15 16 bp between the target sites in order to achieve maximal nuclease activity The target sites can be either 18 or 24 bp in length Use the following image as a reference for the orientation of the binding domains Functional DNA binding domain domain The contribution of individual binding motifs within the DNA binding domain to TAL effector binding efficiency is thought to differ since strong and weak binding motifs exist The A and T binding motifs are thought to fall within the weak binder category while the C and G binding motifs are thought to be strong binders Stretches of more than 5 weak binders should be avoided at the extreme 5 end of the binding domain not counting the 5 N or i
22. ge Chemically competent E coli cells S O C Medium warm to room temperature pUC19 control use as a control for transformation if desired LB plates containing 100 pg mL ampicillin two for each transformation warm at 37 C for 30 minutes 42 C water bath 37 C shaking and non shaking incubator For each transformation aliquot 50 uL of chemically competent E coli cells into a sterile microcentrifuge tube Add 1 uL of the LR recombination reaction from Set up the LR recombination reaction Step 7 previous page into the tube containing 50 uL of competent cells and mix gently Do not mix by pipetting up and down Incubate on ice for 30 minutes Heat shock the cells for 30 seconds at 42 C without shaking Immediately transfer the tubes to ice Add 450 uL of room temperature S O C Medium Cap the tube tightly and shake the tube horizontally 200 rpm at 37 C for 1 hour Spread 20 uL and 100 uL from each transformation on a prewarmed selective plate and incubate overnight at 37 C We generally plate 2 different volumes to ensure that at least 1 plate has well spaced colonies An efficient LR recombination reaction should produce gt 5000 colonies if the entire LR reaction is transformed and plated GeneArt PerfectMatch TALs and GeneArt Precision TALs 13 Analyze transformants Analyze positive clones Analyze transformants by PCR Confirm the expression clone 14 1 Pick 5 colonies and culture them o
23. ing your specific TAL 816010DE 24 Nucleotide Binding Domain containing your specific TAL 816011DE Contents 5 ug of vector DNA lyophilized The TAL vector contains a functional domain and an 18 or 24 nucleotide DNA binding domain Shipping storage All GeneArt PerfectMatch TALs and GeneArt Precision TALs are shipped at room temperature Do not store lyophilized DNA for a prolonged time Upon receipt resuspend the vector and store at 20 C Resuspend the Add 50 uL of distilled water or 10 mM Tris HCl pH 8 0 to the tube containing the vector DNA vector and incubate for one hour at room temperature Resuspend the vector DNA by gently pipetting up and down 5 10 times Store the resuspended vector DNA at 20 C Antibiotic resistance markers are indicated on each tube label The standard delivery amount of DNA is 5 ug GeneArt PerfectMatch TALs and GeneArt Precision TALs 1 Introduction Product information Introduction TAL effectors GeneArt PerfectMatch TALs and GeneArt Precision TALs GeneArt PerfectMatch TALs and GeneArt Precision TALs are optimized to deliver transcriptional effectors to cells in a sequence specific manner These TALs are provided as Gateway adapted entry vectors The sequence is transferred from the entry vector to a destination vector by LR recombination resulting in high level expression of the TAL effectors The GeneArt PerfectMatch TALs are also provided as a CMV expre
24. ith Gateway LR Clonase II Enzyme Mix n Note This step is not required with the CMV vector Transform the recombination reaction into competent E coli cells and select for expression clones e Analyze transformants for the presence of insert by restriction enzyme digestion or colony PCR n Prepare purified plasmid DNA and transfect the cell line of choice E 6 GeneArt PerfectMatch TALs and GeneArt Precision TALs Create a TAL sequence GeneArt PerfectMatch TALs binding site rules The following are guidelines and rules for generating the PerfectMatch TAL sequence The GeneArt PerfectMatch TALs offering allows the construction of TAL effector functional proteins directed to either 18 or 24 base DNA target sites GeneArt PerfectMatch TALs are provided in two types of vectors 1 Gateway adapted entry vector Gateway adapted entry vectors allow easy transfer of target specific TAL domain through a LR recombination reaction into destination vectors designed to facilitate high level expression of the TAL effectors in your cell line of choice A Gateway adapted destination vector is needed for expression plasmid generation Choose a destination vector from our Gateway adapted vector portfolio 2 CMV expression vector mammalian expression vector The mammalian expression vector contains CMV promoter which drives high level expression of the TAL in mammalian systems Per
25. lino and Cathomen 2012 These tools have applications from efficient genomic editing and gene knock out for manipulating the chromosome to modulation of specific promoter activities to allow simple and complex metabolic manipulation in various species of cells The two versions of TALs available are GeneArt PerfectMatch TALs and GeneArt Precision TALs With GeneArt PerfectMatch TALs and GeneArt Precision TALs the researcher can determine the exact DNA loci they would like to have their functionality delivered to and have specific TAL genes built to perform the function The researcher will receive a Gateway adapted entry vector containing the coding sequence for a TAL nuclease or activator designed to bind a specific 18 or 24 base DNA sequence of choice The GeneArt PerfectMatch TALs are also available in a CMV expression vector GeneArt PerfectMatch TALs and GeneArt Precision TALs GeneArt PerfectMatch TALs N TAL Fokl and N TAL Fokl CMV GeneArt PerfectMatch TALs can be designed to target any locus in the genome since there are no restrictions for the 5 base Previously target sites for customized TAL effectors required a 5 T in the target sequences for maximal binding activities The 5 T constraint limited the flexibility of TAL effector target sites in the genome and prevented some specific sites in the genome from being targeted Structure studies suggested the N terminal domain NTD of the TAL effectors not
26. mg mL 10131 035 100 mL 50 mg mL 10131 027 PureLink HiPure Plasmid MiniPrep Kit 25 preps K2100 02 PureLink HiPure Plasmid MidiPrep Kit 25 preps K2100 04 A large selection of Gateway destination vectors are available to facilitate the expression of your gene of interest in virtually any protein expression system For more information about the vectors available refer to our website www lifetechnologies com or contact Technical Support page 28 GeneArt PerfectMatch TALs and GeneArt Precision TALs 27 Technical support Obtaining support For the latest services and support information for all locations go to www lifetechnologies com At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support techsupport lifetech com e Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents e Obtain information about customer training e Download software updates and patches Safety Data Sheets Safety Data Sheets SDSs are available at www lifetechnologies com support SDS Quality Assurance The Quality Assurance Document QAD is a certificate of analysis that provides Document detailed quality control and product qualification informatio
27. ms under 7 C F R 8 340 or any successor regulation c activity directed towards the submission of data generated using a Plant to the United States Department of Agriculture or any equivalent regulatory agency outside of the United States in support of an application for clearance approval or deregulation by such agency d screening or profiling of more than 10 000 distinct compounds high throughput screening e scale up activities the primary focus of which is to increase from small scale to production scale and or g any use of pigs cattle or sheep cells or pig cattle or sheep animals generated using this TALEN product for i agricultural food applications or ii animal models and biomaterials models used for research and development of therapeutic compounds or protocols and production of non cellular products or proteins for therapeutic cosmetic or neutraceutical applications Plant means any eukaryotic plants plant tissues or plant cells including green algae belonging to the plant kingdom defined as Viridiplantae including any progeny propagated through any number of generations or unmodified derivatives of such plants tissues or cells For clarity fungi or blue green algae are specifically excluded from this definition For information on obtaining rights to TALEN technology for any use not permitted herein except use in the therapeutic field and in Plants please contact Life Technologies at outlicensingldlif
28. n the genome Functional DNA binding domain domain The Gateway Technology is a cloning method based on the bacteriophage lambda site specific recombination system which facilitates the integration of lambda into the E coli chromosome and the switch between the lytic and lysogenic pathways Ptashne 1992 In Gateway Technology the components of the lambda recombination system are modified to improve the specificity and efficiency of the system Bushman et al 1985 An LR recombination reaction is performed between the entry clone and the destination vector of choice to generate an expression clone The LR recombination reaction is mediated by LR Clonase II Enzyme Mix a mixture of the bacteriophage A Integrase Int and Excisionase Xis proteins and the E coli Integration Host Factor IHF protein GeneArt PerfectMatch TALs and GeneArt Precision TALs 5 Methods Experimental outline Experimental The table below outlines the steps required to express your GeneArt outline steps PerfectMatch TALs and GeneArt Precision TAL s in cells Step Action Page Determine the sequence of the binding site for your 1 TAL effector protein Synthesize TAL sequence and clone into a Gateway adapted entry vector of choice to generate an entry clone Or clone TAL sequence into the CMV expression vector Perform an LR recombination reaction by mixing the entry clone and the appropriate destination vector w
29. n for each product Certificates of Analysis are available on the disk provided with your clone Limited product Life Technologies Corporation and or its affiliate s warrant their products warranty as set forth in the Life Technologies General Terms and Conditions of Sale found on the Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support 28 GeneArt PerfectMatch TALs and GeneArt Precision TALs References Boch J Scholze H Schornack S Landgraf A Hahn S Kay S Lahaye T Nickstadt A and Bonas U 2009 Breaking the code of DNA binding specificity of TAL type III effectors Science 326 1509 1512 Boch J and Bonas U 2010 Xanthomonas AvrBs3 family type III effectors discovery and function Annu Rev Phytopathol 48 419 36 Bushman W Thompson J F Vargas L and Landy A 1985 Control of Directionality in Lambda Site Specific Recombination Science 230 906 911 Felgner P L Holm M and Chan H 1989 Cationic Liposome Mediated Transfection Proc West Pharmacol Soc 32 115 121 Felgner P L a and Ringold G M 1989 Cationic Liposome Mediated Transfection Nature 337 387 388 Kertbundit S Greve H d Deboeck F Montagu M V and Hernalsteens J P 1991 In vivo Random P glucuronidase Gene Fusions in Arabidopsis thaliana Proc Natl Acad Sci USA 88 5212 521
30. nefit of the buyer The buyer may also use standard molecular biology techniques to make additional copies of this product for purposes of internal research for the sole benefit of the buyer but the buyer may not modify the sequence of the TAL Effector within this product Any buyer that is a for profit entity may not use this product its components or materials or cells made using this product or its components to generate any animal without first obtaining additional rights from Life Technologies The buyer cannot sell or otherwise transfer a this product b its components or c materials cells or organisms made using this product or its components including but not limited to Plants in all cases hereunder to a third party or otherwise use this product its components or materials cells or organisms made using this product or its components for any Commercial Purpose or Development Purpose with the sole exception that buyer may transfer this product its components and or materials cells or organisms made using this product or its components to i the buyer s legal affiliates and or ii a scientific collaborator provided that each such legal affiliate and or scientific collaborator agrees in writing 1 not to transfer such product its components or materials cells or organisms made using such product or its components to any third party and 2 to use such product its components and materials cells and organisms made using such p
31. or sequence 918 2601 Pro Ala Phe Leu Tyr Lys Val Val a LiG TAC AAA GTG GTT C ATG TTT CAC CRA 911 ACAAGTTIGT ACAAAAAAG TOPICRARN A TGTTIT attB1 attB2 10 GeneArt PerfectMatch TALs and GeneArt Precision TALs Perform the LR recombination reaction Introduction Positive control LR Clonase II Enzyme Mix Materials needed Perform an LR recombination reaction between the entry clone and the appropriate destination vector We recommend that you include a positive control see below and a negative control no LR Clonase II Enzyme Mix in your experiment Note This step is not required when using the GeneArt PerfectMatch TAL N TAL FokI CMV The pENTR gus plasmid is used as a positive control for LR recombination and expression Using the pENTR gus entry clone in an LR recombination reaction with a destination vector will allow you to generate an expression clone containing the gene encoding B glucuronidase gus The pENTR gus positive control is supplied with the LR Clonase II Enzyme Mix The LR Clonase II Enzyme Mix is available separately The LR Clonase II Enzyme Mix combines the proprietary enzyme formulation and 5X LR Clonase Reaction Buffer previously supplied as separate components in LR Clonase Enzyme Mix into a single tube format Use the protocol provided on page11 to perform the LR recombination reaction using LR Clonase II Enzyme Mix Note You may perform the LR
32. r localization signal tag It contains 3 amino acids mutated from T TALs It contains 3 amino acids mutated from T TALs NLS Truncated versions of the vector contain the SV40 nuclear localization signal NLS TAL C term C terminus domain of the TAL containing activation domain Fokl Fokl nuclease domain of the TAL Specific N TAL Fokl Fokl CMV vector features The following features are found in the specific GeneArt PerfectMatch TAL vector noted Vector Feature Description N TAL Fokl rrnB T1 and T2 transcription Protects the cloned gene from expression by terminators vector encoded promoters thereby reducing possible toxicity Orosz et al 1991 N TAL Fokl M13 Forward 20 priming site Allows sequencing in the sense orientation N TAL Fokl attL1 and attL2 sites Allows recombinational cloning of the gene of interest from an entry clone Landy 1989 N TAL Fokl Kanamycin resistance gene Allows selection of the plasmid in coli N TAL Fokl CMV Pomy Human cytomegalovirus CMV immediate early promoter enhancer Allows efficient high level expression of TAL Fokl protein N TAL Fokl CMV BGHpA Bovine growth hormone BGH polyadenylation signal Allows efficient transcription termination and polyadenylation of mRNA N TAL Fokl CMV Ampicillin resistance gene C Allows selection of the plasmid in coli C complementary strand 18 GeneArt PerfectMatch TAL
33. re found in the specific GeneArt Precision TALs entry vector features vector noted Vector Feature Description TAL Fokl Fokl Fokl nuclease domain of the TAL TAL vp16 vp64 Vp16 or vp64 Effector domain of the TAL activator TAL KRAB KRAB repressor Effector domain of the TAL TAL MCS MCS Multiple cloning site for insertion of custom effector domains into the TAL TAL MCS Gly Ser linker Flexible peptide linker to prevent steric hindrance between domains GeneArt PerfectMatch TALs and GeneArt Precision TALs 25 Map of pENTR gus entry vector Description Map of control vector 26 pENTR gus is a 3841 bp entry vector containing the Arabidopsis thaliana gene for P glucuronidase gus Kertbundit et al 1991 and is included as a positive control with Gateway LR Clonase II Enzyme Mix Cat nos 11791 020 and 11791 100 The gus gene was amplified using PCR primers containing attB recombination sites The amplified PCR product was then used in a BP recombination reaction with pDONR 201 to generate the entry clone For more information about the BP recombination reaction refer to the Gateway Technology with Clonase I manual The figure below summarizes the features of the pENTR gus vector The complete sequence for pENTR gus is available from our Web site www lifetechnologies com or by contacting Technical Support see page28 Comments for pENTR gus 3841 nucleotides
34. res of GeneArt PerfectMatch TAL Vectors ccscscsesesesesesssesesessssscssssssssssssesesesescsesesesescacsescacseecaeeeees 18 Map of TAL Fok Entry Vector cccccceeceeeeceeeeceeeeeeceneeceaeeeeaeeccaceeseaeeseaeeseaeeseaeeesneeseaeeseaeeseeeseieeeseeessaeeees 19 Map Of AL 1p16 Entry Vector lo lo lee ali lao pal den liado ld aed a 20 Map of TAL vp64 Entry Ve C OT e e a a a a a aa A a a Aaa aee e e Eaa aa inea 21 Multiple cloning site of TAL MCS Entry Vector cccccececceesceeeeceeeeeeeeeeeeeeeeeseaeeeceeeeecaeeseaeeeeeeseneeesseeesseeeees 24 Features of GeneArt Precision TALS entry V CtOIS cccccccsscscsssecsesscecsesecsesecacsesacsesecsesecseseacseecsesecseseeeeses 25 Map ofipENTR gus entry vector accio det A whet eet sheet terested 26 Accessory ProdUcts aia 27 Technical Support iii eee ee la eee eee ee eee 28 REFERENCES iia Ai ts 29 Contents and storage Ordering GeneArt PerfectMatch TALs functional domain Catalog no infopmation Truncated N TAL Foki 816508DE Truncated N TAL Fokl CMV 816509DE GeneArt Precision TALs functional domain Catalog no Native TAL Fokl 816510DE Truncated TAL Fokl 816511DE Native TAL vp16 activator 816512DE Native TAL vp64 activator 816514DE Native TAL MCS 816516DE Truncated TAL MCS 816517DE Native TAL KRAB repressor 816518DE Truncated TAL KRAB repressor 816519DE DNA binding domain Catalog no 18 Nucleotide Binding Domain contain
35. roduct and or its components solely for research as set forth in this limited use label license and not for Commercial Purposes or Development Purposes If this product is subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Mice generated using this product may be donated to The Jackson Laboratory by not for profit buyers and by for profit buyers who have obtained the additional rights noted in the preceding paragraph Please include a copy of this limited use label license with all donations to identify the TAL Effector technology used Commercial Purpose means any activity for consideration including but not limited to a any use directly or indirectly in manufacturing production production of biomolecules or quality control b any use to provide a service information or data for consideration c any use for therapeutic diagnostic or prophylactic purposes or d any sale resale leasing or licensing whether or not for research purposes Development Purpose means any a clinical activity following IND enabling preclinical toxicological studies or equivalents thereof b activity in any agricultural field trial including any such field trial that would be subject to regulation by the United States Department of Agriculture if the plants were to constitute genetically engineered organisms under 7 C F R 8 340 or any successor regulation c activity
36. s and GeneArt Precision TALs Map of TAL Fokl Entry Vector TAL Fokl Entry The map below shows the elements of the TAL FokI Entry Vector The region of Vector map the entry clone corresponding to the TAL is variable depending upon the length of the sequence you ordered The complete sequence for your clone in gb format is available on the disk provided with your clone GeneArt PerfectMatch TALs and GeneArt Precision TALs 19 Map of TAL vp16 Entry Vector TAL vp16 Entry The map below shows the elements of the TAL vp16 Entry Vector The region of Vector map the entry clone corresponding to the TAL is variable depending upon the length of the sequence you ordered The complete sequence for your clone in gb format is available on the disk provided with your clone 20 GeneArt PerfectMatch TALs and GeneArt Precision TALs Map of TAL vp64 Entry Vector TAL vp64 Entry The map below shows the elements of the TAL vp64 Entry Vector The region of Vector map the entry clone corresponding to the TAL is variable depending upon the length of the sequence you ordered The complete sequence for your clone in gb format is available on the disk provided with your clone GeneArt PerfectMatch TALs and GeneArt Precision TALs 21 22 TAL KRAB Entry Vector map Map of TAL KRAB Entry Vector The map below shows the elements of the TAL KRAB Entry Vector The region of the entry clone corresponding to the TAL is vari
37. s legal affiliates and or ii a scientific collaborator provided that each such legal affiliate and or scientific collaborator agrees in writing 1 not to transfer such product its components or materials cells or organisms made using such product or its components to any third party and 2 to use such product its components and materials cells and organisms made using such product and or its components solely for research as set forth in this limited use label license and not for Commercial Purposes or Development Purposes If this product is subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Commercial Purpose means any activity for consideration including but not limited to a any use directly or indirectly in manufacturing production production of biomolecules or quality control b any use to provide a service information or data for consideration c any use for therapeutic diagnostic or prophylactic purposes or d any sale resale leasing or licensing whether or not for research purposes Development Purpose means any a clinical activity following IND enabling preclinical toxicological studies or equivalents thereof b activity in any agricultural field trial including any such field trial that would be subject to regulation by the United States Department of Agriculture if the plants were to constitute genetically engineered organis
38. ssion vector for high level expression in mammalian cells without an LR recombination step Transcription activator like TAL effector proteins are naturally occurring transcriptional activators secreted by Xanthamonas spp into their plant hosts They are injected into plant host cells via a Type III secretion system and travel to the nucleus where they bind to and activate specific promoter sequences that lead to changes that are permissive for bacterial infection Boch and Bonas 2010 TAL effector proteins consist of constant N and C terminal domains containing translocation and nuclear localization activation signals respectively flanking a central repeat domain Each repeat is 34 35 amino acids in length with two centrally located residues that make up a repeat variable domain RVD that dictates the affinity of the repeat for different nucleotide targets Combination and order of various repeat types define the genomic target site specificity of a particular TAL effector The deciphering of this TAL effector code led to the engineering of designer TAL effector proteins that function as a vehicle to target functionality of essentially any open region of the chromosomes of plants bacteria yeast flies and mammalian cells Boch et al 2009 Moscou and Bogdanove 2009 Activities such as activators repressors and nucleases have been demonstrated to be addressable via this powerful system Li et al 2011 Scholze and Boch 2011 Musso
39. the central repeat domain is responsible for the interaction with the 5 T of the target We developed our second generation TALs GeneArt PerfectMatch TALs by mutating the N terminal domain to reduce its specificity for 5 T GeneArt PerfectMatch TALs can target DNA sequences with any 5 base T G C or A with performance comparable to that of GeneArt Precision TALs GeneArt PerfectMatch TALs contain a truncated TAL engineered with FokI nuclease The FokI TAL nuclease pair binds to duplex DNA at the target sites designated by the DNA binding domains to cleave the DNA There are two versions of GeneArt PerfectMatch TALs e N TALFokI a Gateway adapted entry vector which allows easy transfer through a LR recombination reaction to destination vectors designed to facilitate high level expression of the TAL effectors in your cells of choice e N TAL FokI CMV a CMV expression vector which contains a CMV promoter to drive high level expression of the TAL in mammalian systems It can be directly used without extra subcloning Functional DNA binding domain domain GeneArt PerfectMatch TALs and GeneArt Precision TALs 3 GeneArt Precision TALs TAL Fokl TAL VP16 and TAL VP64 TAL KRAB Unlike GeneArt PerfectMatch TALs Precision TALs have a conserved T binding motif at the N terminus of the TAL effector protein and so require a 5 T for maximal binding activity GeneArt Precision TALs are available as the
40. vernight in LB or SOB medium containing 100 pg mL ampicillin 2 Isolate plasmid DNA using your method of choice We recommend using the PureLink HiPure Plasmid MiniPrep Kit Cat no K2100 02 or the PureLink HQ Mini Plasmid Purification Kit Cat no K2100 01 See Additional products p27 3 Analyze the plasmids by restriction analysis to confirm the presence of the insert You can also analyze positive transformants using PCR For PCR primers use a primer that hybridizes within the vector e g T7 Promoter Primer Catalog no N560 02 and one that hybridizes within your insert You will have to determine the amplification conditions If you are using this technique for the first time you may want to perform restriction analysis in parallel Artifacts may be obtained because of mispriming or contaminating template The protocol below is provided for your convenience Other protocols are suitable Materials needed PCR SuperMix High Fidelity Cat no 10790 020 Appropriate forward and reverse PCR primers 20 uM each Note To avoid PCR errors due to highly repetitive sequences we recommend designing primers that hybridize to the N terminal domain of the TAL sequence Procedure 1 For each sample aliquot 48 uL of PCR SuperMix High Fidelity into a 0 5 mL microcentrifuge tube Add 1 uL each of the forward and reverse PCR primer 2 Pick 5 colonies and resuspend them individually in 48 uL of the PCR SuperMix remember to make a
Download Pdf Manuals
Related Search