Cellecta User Manual, Cloning of shRNA Templates into
Contents
1. The laws of the State of California shall govern the interpretation and enforcement of the terms of these Licenses The Buyer End User acknowledges that Product has been developed by Cellecta based on licenses from Third Parties and agrees with the Terms of Limited Use for the Buyer End User provided by the Third Parties Life Technologies Corporation End User Label License for the use of Lentiviral Expression System This product or service based upon the Lentiviral Expression System is sublicensed from Life Technologies Corporation under U S Patent Nos 5 686 279 5 834 256 5 858 740 5 994 136 6 013 516 6 051 427 6 165 782 6 218 187 6 428 953 6 924 144 7 083 981 and 7 250 299 and corresponding patents and applications in other countries for internal research purposes only Use of this technology for gene therapy applications or bioprocessing other than for nonhuman research use requires a license from GBP IP LLC Please contact GBP IP LLC 537 Steamboat Road Suite 200 Greenwich CT 06830 Use of this technology to make or sell products or offer services for consideration in the research market requires a license from Life Technologies Corporation 5791 Van Allen Way Carlsbad CA 92008 Evrogen IP JSC End User Label License for the use of lentiviral shRNA constructs comprising TagRFP encoded gene This product is for internal non commercial research use only No rights are conveyed to modify or clone the gene encoding fl2. phage viruses cells or tissues created directly or indirectly from the Product or any portion is prohibited without prior written approval separate licenses are available for non research use or applications The Product is not to be used for human diagnostics or included used in any drug intended for human use Care and attention should be exercised in handling the Product by following appropriate research laboratory practices Cellecta s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price Cellecta s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty Cellecta does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned Cellecta disclaims any and all responsibility for injury or damage that may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Purchasers may refuse these licenses by returning the Product unused By keeping or using the enclosed materials you agree to be bound by the terms of these licenses
3. Cellecta Inc 320 Logue Ave Mountain View CA 94043 CELLECTA Ht covets E Cloning of shRNA Templates into shRNA Expression Vector User Manual Table of Contents v 4 Background Required Materials Cloning of shRNA Template Oligonucleotides into shRNA Expression Vector Identify clones with the target shRNA template Purify Plasmid shRNA Construct Technical Support Safety Guidelines References rammMooW DY NOM KR RWYN HB e A Background The protocols below provide the instructions on how to phosphorylate shRNA template oligos ligate them to shRNA cloning vectors transform competent cells and grow plasmid DNA to generate plasmid shRNA expression constructs For packaging of constructs into pseudovirus please refer to the User Manual for Packaging and Transduction of Lentiviral Constructs Please read the entire user manual before proceeding with your experiment B Required Materials For Phosphorylation and Annealing of shRNA Template Oligonucleotides e T4 Polynucleotide Kinase and 10X reaction buffer Recommended New England BioLabs T4 Polynucleotide Kinase 10 U ul Cat MO201S e rATP Recommended GE Amersham Cat 27 2056 01 For Ligating and Transforming shRNA Constructs e Linearized shRNA Expression Vector NOTE For DECIPHER pRSI9 U6 sh UbiC TagRFP 2A Puro Empty Vector undigested Cellecta recommends digestion with Fermentas Bpil Bbsl 10 U ul Cat ER1012 and gel purificat
4. e expression lentivectors pVSV G expression vector or any other vector to prevent generation of recombinant replication competent pseudovirus e None of the HIV 1 genes gag pol rev will be present in the packaged pseudoviral genome as they are expressed from packaging plasmids lacking packaging signal therefore the lentiviral particles generated are replication incompetent e Pseudoviral particles will carry only a copy of your expression construct Despite the above safety features use of HIV based vectors falls within NIH Biosafety Level 2 criteria due to the potential biohazard risk of possible recombination with endogenous viral sequences to form self replicating pseudovirus or the possibility of insertional mutagenesis For a description of laboratory biosafety level criteria consult the Centers for Disease Control Office of Health and Safety Web site at http www cdc gov od ohs biosfty bmbl4 bmbl4s3 htm It is also important to check with the health and safety guidelines at your institution regarding the use of lentiviruses and follow standard microbiological practices which include e Wear gloves and lab coat at all times when conducting the procedure e Always work with pseudoviral particles in a Class II laminar flow hood e All procedures are performed carefully to minimize the creation of splashes or aerosols e Work surfaces are decontaminated at least once a day and after any spill of viable material e All cultur
5. es stocks and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving Materials to be decontaminated outside of the immediate laboratory area are to be placed in a durable leakproof properly marked biohazard infectious waste container and sealed for transportation from the laboratory Tech Support tech cellecta com 5 of 6 v 3 2 7 11 Cellecta Inc 320 Logue Ave Mountain View CA 94043 CELLECTA Ht covets E H References Lentiviral delivery vector reviews Curran MA Nolan GP Nonprimate lentiviral vectors Curr Top Microbiol Immunol 2002 261 75 105 Curran MA Nolan GP Recombinant feline immunodeficiency virus vectors Preparation and use Methods Mol Med 2002 69 335 50 Loewen N Barraza R Whitwam T Saenz DT Kemler I Poeschla EM FIV Vectors Methods Mol Biol 2003 229 251 71 Naldini L Lentiviruses as gene transfer agents for delivery to non dividing cells Curr Opin Biotechnol 1998 Oct 9 5 457 63 I Limited Use License Agreement Cellecta Inc Limited License Cellecta grants end users of the Product a non transferable non exclusive license to use the reagents for internal research use only In all cases sale or other transfer or distribution to third parties of i the Product or any portion ii DNA RNA nucleotide sequences of shRNA and shRNA constructs or libraries created from the Product or any portion or of iii transformed
6. ides into shRNA Expression Vector 1 Phosphorylate and Anneal the shRNA Template Oligonucleotides gt Oa 0 g Note This protocol was developed for regular non phosphorylated oligos If your oligonucleotides are already phosphorylated dilute them to 10 uM in 1X T4 polynucleotide kinase buffer heat at 95 C for 2 min and anneal as in steps 1 d 1 e below Dissolve the shRNA template oligonucleotides in an appropriate amount of deionized water to a final concentration of 20 uM Set up 20 ul phosphorylation annealing reactions for each shRNA template ul Top Strand shRNA template oligo 20 uM ul Bottom Strand shRNA template oligo 20 uM ul 10X T4 Polynucleotide Kinase Buffer ul 5 mM ATP 13 ul Deionized water 1 ul T4 Polynucleotide Kinase 10 U ul 20 ul Total volume N Ne he For the insert minus control use 2 ul deionized water in place of the top and bottom strands Incubate the phosphorylation reaction at 37 C for 30 minutes in a thermocycler Heat the reaction mix to 95 C for 2 min in a thermocycler Turn off the thermocycler and let it cool to room temperature Take a 2 ul aliquot from the reaction 1 uM shRNA template and make a 1 5 dilution by adding 8 ul of 1X T4 Kinase buffer Mix well Use 0 5 ul of the diluted shRNA template 0 2 uM for the following ligation reaction 2 Ligate the shRNA Template into Linearized shRNA Lentivector d Set up 10 ul ligation reactions for each phosphorylated shRNA tem
7. ion using QIAGEN QIAquick Gel Extraction Kit Cat 28706 e T4 DNA Ligase and 10X ligation reaction buffer Recommended New England BioLabs T4 DNA Ligase 400 U ul Cat MO202S Before using dilute T4 DNA ligase 10 fold with 1X T4 DNA ligase buffer to 40 U ul e Competent E coli cells RecA Recommended Invitrogen OmniMAX 2 T1 cells Cat C8540 03 e Petri plates containing LB Agar media with 100 ug ml Ampicillin or Carbenicillin For Screening shRNA Inserts e Taq DNA polymerase and 10X reaction buffer Recommended BDB Clontech Titanium Taq DNA polymerase Cat 639208 e dNTP mix Recommended GE Amersham dNTP set Cat 27 2035 01 e Thermal Cycler Tech Support tech cellecta com 1 of 6 v 3 2 7 11 Cellecta Inc 320 Logue Ave Mountain View CA 94043 CELLECTA Ht covets E 3 1X TAE Agarose gel For Purifying shRNA Constructs after Cloning Plasmid purification kit Recommended QIAGEN Endotoxin free Plasmid Kit The following kit combinations can be used for Midi scale preparation of endotoxin free DNA gt QIAfilter Plasmid Midi Kit Cat 12243 and EndoFree Plasmid Maxi Kit Cat 12362 gt QIAfilter Plasmid Midi Kit Cat 12243 and EndoFree Plasmid Buffer Set Cat 19048 Please visit the QIAGEN website to download the specialized protocol that is not contained in the user manual gt http wwwi1 qiagen com literature protocols pdf QP15 pdf C Cloning of shRNA Template Oligonucleot
8. nue to grow the culture for another 6 hours c Store the bacterial culture at 4 C 2 Screen for shRNA template inserts a Prepare a PCR master mix for each clone you would like to screen for the presence of an shRNA template insert as follows i rxn 10 rxn Composition 0 5 ul 5 ul Forward PCR Primer 10 uM 0 5 ul 5 ul Reverse PCR Primer 10 uM 0 5 ul 5 ul 50X dNTP mix 10 mM of each 2 5 ul 25 ul 10X PCR Reaction Buffer 19 5 ul 195 ul Deionized water 0 5 wl 5 ul Taq DNA Polymerase 5 U ul 24 0 ul 240 ul Total volume b Mix the master mix very well and aliquot 24 ul into each well of a 96 well PCR plate or individual tubes c Add 1 ul of each bacterial culture from B 1 into each well or tube from B 2 b Mix Proceed with PCR using the following program 94 C 4 min 1 cycle 94 C 0 5 min then 68 C 1 min 25 cycles 68 C 2 min 1 cycle e Take 5 ul of PCR product from step d and run it on a 3 agarose EtBr gel in 1X TAE buffer f Confirm identity of shRNA template inserts by sequence analysis of positive PCR products using the Forward PCR primer Tech Support tech cellecta com 3of6 v 3 2 7 11 Cellecta Inc 320 Logue Ave Mountain View CA 94043 CELLECTA BH cavers E E Purify Plasmid shRNA Construct a Take 15 ul of each positive bacteria culture from Step B 1 c inoculate each clone in 10 mli of LB broth media with 100 ug ml ampicillin or carbenicillin and grow overnight at 37 C with Shaking b P
9. plate as follows 1 0 ul Linearized shRNA Expression Vector 10 ng ul see Required Materials 0 5 ul Phosphorylated ds shRNA template step 1 0 2 uM 1 0 ul 10X T4 DNA Ligase Buffer 6 5 ul Deionized water 1 0 ul T4 DNA ligase 40 U ul 10 0 ul Total volume Tech Support tech cellecta com 2of6 v 3 2 7 11 Cellecta Inc 320 Logue Ave Mountain View CA 94043 CELLECTA Ht covets E For negative control use insert minus Dilute T4 DNA ligase 400 U ul 10 fold to 40 U ul with 1X T4 DNA ligase buffer if you are using New England Biolabs enzyme b Incubate the ligation reaction at 16 C for 1 2 hrs 3 Transform E coli with the ligation product a For each shRNA template use the whole volume of ligation product for transformation b Follow the manufacturer s protocol for transforming the competent cells Plate an appropriate amount of cells on LB plates with 100 ug ml ampicillin or carbenicillin and grow overnight at 37 C d You could expect to get at least 10 fold more colonies in experimental samples in comparison with negative control vector only ligation reaction D Identify clones with the target shRNA template 1 Prepare colony cultures a Randomly pick up 10 well separated colonies from each plate and grow each clone in 100 ul of LB Broth with 100 ug ml ampicillin or carbenicillin at 37 C for 2 hours with shaking b Take 1 ul of each bacteria culture for PCR screening see B 2 and conti
10. uorescent protein contained in this product The right to use this product specifically excludes the right to validate or screen compounds For information on commercial licensing contact Evrogen Licensing Department email license evrogen com Tech Support tech cellecta com 6 of 6 v 3 2 7 11
11. urify shRNA lentivector construct plasmid DNA in Midi or Maxi scale using an Endotoxin free plasmid purification kit F Technical Support For additional information or technical assistance please call or email us at Phone 650 938 3910 877 938 3910 Toll Free Fax 650 938 3911 E mail General Information info cellecta com Technical Support tech cellecta com Sales sales cellecta com For more information about Cellecta s products and services please visit our web site at http www cellecta com Cellecta Inc 320 Logue Ave Mountain View CA 94043 Tech Support tech cellecta com 4 of 6 v 3 2 7 11 Cellecta Inc 320 Logue Ave Mountain View CA 94043 CELLECTA Ht covets E G Safety Guidelines The HIV based lentivector system is designed to maximize its biosafety features which include e A deletion in the enhancer of the U3 region of 3 ALTR ensures self inactivation of the lentiviral construct after transduction and integration into genomic DNA of the target cells e The RSV promoter upstream of 5 LTR in the lentivector allows efficient Tat independent production of viral RNA reducing the number of genes from HIV i that are used in this system e Number of lentiviral genes necessary for packaging replication and transduction is reduced to three gag pol rev The corresponding proteins are expressed from different plasmids lacking packaging signals and share no significant homology to any of th
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