GenTarget`s EcoTMPlasmid DNA Miniprep Kit User Manual
Contents
1. Each vector contains a multiple cloning site MCS region for sub cloning of any Promoter Target expression cassette or promoter only insert depending on vector types These vectors do not contain any promoters ahead of the MCS and are ideal for promoter studies They contain a selection marker under the RSV promoter for selection and monitoring These lentivectors were engineered with several key genetic elements WPRE cPPT RRE for producing high titer expression lentivirus See vector schematic maps below In order to produce expression lentiviral particles lentivirus the lentivector must be co transfected with a packaging plasmid mixture CAT HT Pack into the virus production 293T cell line after incubation the virus containing supernatant is collected GenTarget s lentivectors are fully compatible with most packaging mixtures on the market GenTarget also provides packaging plasmid mixture Cat HT pack and 293T cells Cat TLV C Promoter less lentiviral vector product manual Page 1 of 10 www gentarget com GenTarget Inc 2015 G 7930 Arjons Drive Suite B San Diego CA 92126 el arae ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Each vector contains an ampicillin maker and can be propagated in LB medium containing 100 wg ml of ampicillin The full vector sequence was verified by sequence analysis and can be downloaded from our website Note Unwanted recombination can2. occur during lentivector propagation due to the lentivectors LTR arms therefore we recommend verifying your propagated lentivector by restriction digestion Product Contents Each vector is provided as 6 0 wg per vial x 1 vial lyophilized This amount is sufficient for 10 to 20 cloning processes To reconstitute the dried vector simply add 10 20 ul of DNAse free water to obtain the desired vector concentration and gently vortex The plasmid can be propagated if more DNA is needed for another purpose Key Features e An MCS region containing multiple Restriction Enzyme sites for sub cloning of a promoter target cassette e In some vectors a commonly used reporter GFP RFP or luciferase is pre installed after the MCS region the reporter can be used in a sub clone of any promoter only inserts e A variety of antibiotic selection markers is available including fluorescent antibiotic fusion dual markers for convenient tracing of viral performance e Engineered lentivector backbone for high titer virus production eA modification mutation in the 3 UTR region SIN feature which produces only replication incompetent lentivirus for enhanced Bio safety e These lentivectors are fully compatible with most packaging mixtures on the market Product schematic maps 1 Cat LV PLI1 Promoter less lentiviral vector product manual Page 2 of 10 www gentarget com GenTarget Inc 2015 G 7930 Arjons Drive Suite B Sa
3. G 7930 Arjons Drive Suite B San Diego CA 92126 el arae ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Promoter less Lentiviral Expression Vector Cat Product Name Amounts LV PL1 Promoterless MCS lentivector Bsd 6 ug lyophilized plasmid DNA LV PL2 Promoterless MCS lentivector Bsd RFP Ug lyophilized plasmid DNA LV PL3 Promoterless MCS lentivector GFP Bsd 6 ug lyophilized plasmid DNA LV PL4 Promoterless MCS lentivector Puro 6 ug lyophilized plasmid DNA LV PL5 Promoterless MCS lentivector Puro RFP Ug lyophilized plasmid DNA LV PL6 Promoterless MCS lentivector Neo 6 ug lyophilized plasmid DNA Storage Store at 20 C after reconstituted with DNAase free water Stable for gt 12 months Product Description GenTarget s lentivector system is Human Immunodeficiency Virus 1 HIV based plasmids for gene expression and knockdown The lentivectors are used to generate lentiviral particles lentivirus that can be transduced into almost all kinds of mammalian cells including stem cells primary cells and non dividing cells both in vivo and in vitro Lentiviral Particles stably integrate into the transduced cells genome for long term expression making it a great gene transfer agent GenTarget provides several Promoterless expression vectors containing a variety of antibiotic markers or fluorescent antibiotic fusion dual markers
4. agent such as LF2k from Invitrogen FuGene from Roche or your preferred transfection reagent Follow the packaging plasmid manual for the amount of DNA to use Day 2 or 3 e Harvest lentiviral supernatant 48 72 hours after transfection Centrifuge 5 minutes at 1500 rpm to remove cell debris and filter at 0 22 um e Supernatants can be directly used simply add into any cell culture for expression assay Depending upon cell type the expression will peak after 72 hours to one week If the lentivector contains a fluorescent marker you can monitor the virus performance by visualizing the fluorescent signal at 48 72 hours post infection e Store supernatants long term at 80 C in aliquots avoid repeated freeze thaw cycles Promoter less lentiviral vector product manual Page 7 of 10 www gentarget com GenTarget Inc 2015 G 7930 Arjons Drive Suite B San Diego CA 92126 el arae ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Warranty and purchase terms 1 This product is warranted to meet its quality as described when used in accordance with its instructions GenTarget disclaims any implied warranty of this product for any particular application In no event shall GenTarget be liable for any incidental or consequential damages in connection with this product GenTarget s sole remedy for breach of this warranty should be at GenTarget s option to replace the product 2 The passing giving re pr
5. amHI Clal Spel Nhel BsrGI Nhel Promoter less lentiviral vector product manual Page 4 of 10 www gentarget com GenTarget Inc 2015 G 7930 Arjons Drive Suite B San Diego CA 92126 el arae ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com 5 Cat LV PLS BamHI Ie MCS RSV BstZ171 e Clal Apal Spel Sall LV PL1 L Nhel 5 LTR 7088 bp J i Xbal FF sir A BsrGI Puro RFP sf AmpR MCS sequence at vector position 1 54 in product LV PLS5 GGATCC ATCGAT ACTAGT CTAGC GTACA BamHI Clal Spel Nhel BsrGI 6 Cat LV PL6 BamHI lt MCS Sv40Pro BstZ171 i Clal Apal Spel Sall Nhel Xbal BsrGI Nhel i LV PL1 5 LTR 7088 bp t LTRA I F SV40pA MCS sequence at vector position 1 67 in product LV PL6 GGATCC ATCGAT ACTAGT CTAGC GTACAAAGTGGTGCTAGC BamHI Clal Spel Nhel BsrGI Nhel Promoter less lentiviral vector product manual Page 5 of 10 www gentarget com GenTarget Inc 2015 G 7930 Arjons Drive Suite B San Diego CA 92126 el arae ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com The Promoterless lentiviral expression vector is designed to help you create a lentivirus to deliver and express a gene of interest from a promoter of your choice The first step is to sub clone the promoter into the lentivector We strongly recommend that users possess a working knowledge of molecular cloning techniques and be familiar wi
6. e lentivirus expressing LoxP GFP Stop LoxP RFP ColorSwitch cassette used to monitor the CRE recombination event in vivo CRISPR hu Preamde lentivirus express humanzied wild type Cas9 CAS9 endonuclease for genomic editing with CRISPR TetR Premade lentivirus expressin TetR tetracycline regulator protein inducible the repressor protein for the inducible expression system expression repressor Premde lentivirus for human and mouse iPS Myc NANOG iPS factors OCT4 SOX2 FLF4 factors with different fluorescent and antibitoic markers T antigen Express SV40 large T antigen with different selection markers Expression Cell Premade lentivirus for cell organelle imaging The fluorescent Organelle marker GFP RFP CFP was sub cellular localized in different imaging cell organelle for living cell imaging Lacz Express different full length B galactosidase lacZ with different expression selection markers Anti miNA Pre made lentivirus expression a specific anti miRNA cassette lentivirus Fluorescent Pre made lentivirus expression a GFP RFP CFP ORF fusion ORF fusion target Pre made Premade shRNA lentivirus for knockdown a specific genes P53 shRNA LacZ Luciferase and more lentivirus microRNA Premade lentivirus expression human or mouse precursor and anti miRNA And anti miRNA lentivector and virus for human and microRNA mouse miRNA Promoter less lentiviral vector product manual Page 9 of 10 www gentarget com GenTarget I
7. n Diego CA 92126 el arae ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com MCS RSV Se d LV PL1 5 LTR 7088 bp l LTR Sv40pA ra AmpR MCS sequence at vector position 1 67 in product LV PL1 GGATCC ATCGAT ACTAGT CTAGC GTACAAAGTGGTGCTAGC BamHI Clal Spel Nhel BsrGI Nhel 2 Cat LV PL2 BamHI BstZ171 Clal Apal Spel Sall Nhel Xbal BsrGI Nhel Xbal MCS RSV b _Bsd RFP l LV PL1 5LTR _ 7088 bp t LTR A SV40pA Z AmpR MCS sequence at vector position 1 82 in product LV PL2 GGATCC ATCGAT ACTAGT CTAGC GTACAAAGTGGTGCTAGC BamHI Clal Spel Nhel BsrGI Nhel in of Promoter less lentiviral vector product manual Page 3 of 10 www gentarget com GenTarget Inc 2015 G 7930 Arjons Drive Suite B San Diego CA 92126 el arae ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com 3 Cat LV PL3 BamHI Ie MCS RSV BstZ17I Clal Apal Spel Fsel LV PL1 L Smal 5 LTR 7088 bp F LTR A _GFP Bsd Xbal Sall Ve AmpR MCS sequence at vector position 1 54 in product LV PL3 GGATCC ATCGAT ACTAGT GGG BamHI Clal Smal TCGAC Sall 4 Cat LV PL4 BstZ171 Clal Apal Spel Sall Nhel Xbal BsrGI Nhel LV PL1 5 LTR 7088 bp t LTRA a SV40pA AmpR MCS sequence at vector position 1 67 in product LV PL4 GGATCC ATCGAT ACTAGT CTAGC GTACAAAGTGGTGCTAGC B
8. nc 2015 7930 Arjons Drive Suite B San Diego CA 92126 el arae ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com lentivirus Negative Premade negative control lentivirus with different markers control serves as the negative control of lentivurs treatment for lentiviruses validation of the specificity of any lentivirus target expression effects Other Ready to use lentivirus expressing specific enzymes with Enzyme different selection markers expression Promoter less lentiviral vector product manual Page 10 of 10 www gentarget com GenTarget Inc 2015
9. oducing or re selling of this product is prohibited without written permission from GenTarget Inc 3 This product is intend for research use only not for therapeutic or diagnostic uses 4 By paying the purchase price the buyer is granted a non transferable non exclusive license to use the product This product is limited to the laboratory that the product is delivered to Furthermore research use only means that this product is excluded without limitation from resale repackaging or modification to be used for the making or selling of any commercial product s or services without the written approval of GenTarget You may contact our Business Development department at support gentarget com for product proprietary information Safety Precaution Gentarget lentivectors adapt must advanced lentiviral safety features using the third generation vectors with self inactivation SIN 3UTR and the generated lentivirus is replication incompetent However please use extra caution when using lentiviral particles Use the lentiviral particles in Bio safety II cabinet Ware glove all the time at handling Lentiviral particles Please refer CDC and NIH s guidelines for more details regarding to safety issues References e NIH Guidelines for Biosafety Considerations for Research with Lentiviral Vectors Link e CDC guidelines for Lab Biosafety levels Link e J Virol 2004 78 1421 30 e J Virol 2000 November 74 22 10778 10784 Wa
10. rranty and purchase terms This product is for research use only It is warranted to meet its quality as described when used in accordance with its instructions GenTarget disclaims any implied warranty of this product for particular application In no event shall GenTarget be liable for any incidental or consequential damages in connection with the products GenTarget s sole remedy for breach of this warranty should be at GenTarget s option to replace the products Promoter less lentiviral vector product manual Page 8 of 10 www gentarget com GenTarget Inc 2015 elated GenTarset inc 7930 Arjons Drive Suite B San Diego CA 92126 Phone 858 6788683 Fax 800 3804198 Email orders gentarget com roducts GenTarget s premade lentivirus products Product Product Description Category please click category name to see product s pages Human Premade lentivirus expressin a human mouse or rat gene with mouse or rat RFP Blastididin fusion dual markers ORFs Fluorescent Preamde lentivirus express human codon optimized fluorescent markers protein GFP RFP CFP BFP YFP Luciferase Premade lentivirus for all kinds of luciferase protein expression expression firefly and Renilla with different antibiotic selection markers CRE Premade lentivirus for expressing nuclear permeant CRE recombinase recombinase with different flurescent and antibiotic markers LoxP Premad
11. th restriction enzyme based sub cloning methods as well as the lentivirus production procedure Once the lentivector is made you will need to co transfect it with lentiviral packaging plasmids into 293T cells The packaging plasmids and 293T cells are not included with this product but you may purchase them from GenTarget NOTE Carry out the virus production procedure according to the packaging plasmid manual Sub cloning outline Lentivector preparation e Design and select compatible restriction enzyme sites between the insert promoter of interest and promoterless lentivector e Open the promoterless lentivector at its MCS region by restriction enzyme cutting and dephosphorylate the vector open ends by CIP or another phosphatase Insert preparation e Select the promoter target of your choice e Cut the insert by the same restriction enzymes or overhung compatible enzymes as used in the lentivector preparation above e If the promoter and the target are sub cloned separately in two steps make sure that your promoter contains all necessary components such as TATA box enhancer and a transcription initiation site and that the promoter region does not contain ATG to avoid unwanted transcription start NOTE if the promoter and the target are sub cloned in two steps you have to sequentially open the lentivector in two position to accommodate the promoter and the target T4 ligation ligate the promoter into the lentivec
12. tor Plate the Colonies Promoter less lentiviral vector product manual Page 6 of 10 www gentarget com GenTarget Inc 2015 G 7930 Arjons Drive Suite B San Diego CA 92126 el arae ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com e Transform 1 3 ul ligation mixture into cloning competent cells such as DH5a Top10 or NovaBlue e Spread cells onto an LB agar plate containing 100 ug ml ampicillin Screen the positive colonies e Pick a few colonies and confirm those that are positive by restriction digestion of the purified plasmid DNAs e The insert promoter region may be verified by sequencing analysis Lentivirus production To obtain expression ready lentivirus the generated expression lentivector must be co transfected with packaging plasmid mixture Cat HT pack into 293T cells Cat TLV C GenTarget s lentivector is fully compatible with other vendors packaging plasmids for virus production and can be packaged with either the 2nd or the 3rd packaging system The packaging plasmids and 293T cells are not included in this product but they are available separately from GenTarget or may be obtained from other vendors Lentivirus production outline Day 0 e One day before transfection plate sufficient 293T cells to achieve 70 80 confluence on the day of transfection Day 1 Co transfect the expression lentivector and the packaging plasmid mixture into 293T cells using Transfection Re
Download Pdf Manuals
Related Search