pdsipher and pdsipher -GFP shrna Vector User s Guide
|
|
- Winifred Bishop
- 6 years ago
- Views:
Transcription
1 pdsipher and pdsipher -GFP shrna Vector User s Guide NOTE: PLEASE READ THE ENTIRE PROTOCOL CAREFULLY BEFORE USE Page 1. Introduction Vector Overview Vector Maps 2 4. Materials Provided Additional Materials and Equipment Required Cloning shrna Template Into pdsipher Vectors Transfection Protocol Selection of Plasmid Using Neomycin Technical Support Introduction The plasmid pdsipher is a 3.2 kb vector designed for expression of short hairpin RNA (shrna) in cultured cells following transfection. This shrna is approximately 60 nt long and is designed to form a hairpin structure that mimics 21 nt double stranded small interfering RNA (sirna) that induces RNA interference (RNAi) in gene knockdown experiments. The shrna is expressed from a human H1 promoter by endogenous polymerase III, resulting in high levels of expression of the shrna with a well-defined 3' end resulting in a UU overhang compatible with sirna design. Following successful transfection and expression of the shrna, it is processed by Dicer, the nuclease responsible for producing endogenous sirnas, and is processed into a form recognizable by the complex molecular machinery responsible for RNAi, known as the RISC complex. If the sirna has been designed with sufficient care, the RISC complex will then efficiently degrade RNA corresponding to the designed shrna. To design sirna and shrna sequences for your particular gene we recommend MoleculA s target design services (see for further details). 2 Vector Overview The plasmid pdsipher is 3.2 kb in size and contains many features suitable for shrna cloning, transfection and expression. A human H1 polymerase III promoter for efficient expression of the ~60 bp shrna with BamHI/HindIII cloning sites (bases ) for easy cloning of shrna constructs. Kanamycin/Neomycin dual resistance for both bacterial and mammalian selection (bases ). In addition, pdsipher -GFP contains a green fluorescent protein (GFP) under the control of a CMV promoter so cell transfection efficiencies can easily be assayed. MoleculA pdsipher technical manual January 2005
2 3 Vector Maps pdsipher (cat. #RI-500) Features Feature Position F1 origin SV40/Amp promoter Kan/Neo resistance TK p(a) site puc origin H1 pol. III promoter pdsipher -GFP (cat. #RI-550) Features Feature Position F1 origin SV40/Amp promoter Kan/Neo resistance TK p(a) site puc origin SV40 pa site GFP CMV promoter H1 pol. III promoter For the full sequence of these vectors, please visit 4 Materials Provided 10 µg lyophilized plasmid DNA Technical manual Storage Conditions pdsipher should be stored at -20 o C. Properly stored, product is guaranteed for one year from date of receipt. Safety Warning: To the present knowledge of the supplier, the materials supplied (listed above) do not contain any hazardous ingredients. However, all materials may present some unknown hazard and should be handled with caution. Some of the additional materials required (listed below) may present hazards and it is the sole responsibility of the user to determine what these hazards are and to take appropriate action. MoleculA pdsipher technical manual January 2005
3 5 Additional Materials and Equipment Required The following additional materials are required in order to use pdsipher and pdsipher - GFP to express shrna within cells. Cloning of cassette into pdsipher /pdsipher -GFP: BamHI HindIII Competent cells Annealed oligos corresponding to the shrna transcription cassette (see below for more details). Transfection of plasmid into cells: Cells at approximately 50-70% confluency Serum-Free Media Fetal Bovine Serum (FBS) Maxfect (MoleculA cat #TR ), or other DNA transfection reagent 6 Cloning shrna Template Into pdsipher Vectors 1) Design your sirna sequence. Different sirna sequences will knock down your gene of interest to different levels, due to only a small fraction of the RNA target being accessible for binding and interaction by sirna. We recommend that you design your sirna sequence using MoleculA s target design services. 2) Design shrna inserts for cloning into pdsipher vectors. This needs the following features: BamHI overhang for ligation into the vector. 19 nt sense sirna sequence. Loop sequence. 19 nt antisense sirna sequence. A six (6) T nt polymerase III termination sequence. HindIII overhang for ligation into the pdsipher vector. To design oligos with these features, please visit for our free shrna oligo design tool. 5' GATCC 3' G antisense TTCAAGAGA antisense TTTTTTA 3' antisense AAGTTCTCT antisense AAAAAATTCGA 5' 3) Order two oligos matching the above sequences. PAGE purification is recommended, however, many successful experiments have used standard desalted oligos. 4) Anneal the oligos. Set up the following reaction: Top-strand oligo (1 µg/µl) 1 µl Bottom-strand oligo (1 µg/µl) 1 µl 20X SSC 1 µl dh 2 O 17 µl Heat the reaction (95 C, 10 min) and place at room temperature (60 min). 5) Cut the vector with BamHI and HindIII. Run on a 1% agarose gel and gel purify the band. 6) Ligate the insert and vector together using standard DNA ligase protocols. 7) Transform the ligation into competent cells using protocols for that competent cell line. 8) Check that plasmids resulting from any colonies contain the insert, either by PCR or by restriction digest of the resulting plasmid(s). MoleculA pdsipher technical manual January 2005
4 7 Transfection Protocol - Using Maxfect For each cell line the protocol should be optimized according to the instructions shipped with Maxfect. Please ensure your transfection protocol is suitable for your cell line. The protocol listed here is for 6 well culture plate using Maxfect and should be optimized depending on cell type, well size, and transfection reagent. Although the protocol given here is for Maxfect, other lipid-based transfection reagents can be used with their respective protocols. 1) In a six well or 35mm tissue culture plate, seed ~2*10 5 cells per well in 2ml of D-MEM with 10%FBS. Incubate the cells at 37 C in a CO2 incubator until the cells are 50-70% confluent. This will usually take h. 2) Prepare the following solutions: Solution A: For each transfection, dilute 1µg pdsipher vector into 100µl serum-free or low protein medium without antibiotics. Solution B: For each transfection, dilute 2-8µl of lipid reagent into 100µl serum-free or low protein medium without antibiotics. Peak activity should be at about 6µl. 3) Add the pdsipher vector solution directly to the dilute lipid using a pipet. Mix gently by pipeting the solution and incubate the mixture 20 minutes at room temperature. 4) Wash the cells once with 2ml of low protein or serum-free and antibiotics free medium. 5) For each transfection, add 0.8ml of low protein or serum-free and antibiotics free medium to each tube containing the vector:lipid complex. Mix gently and overlay the diluted complex solution onto the washed cells. 6) Incubate the cells 5-7 h at 37 C in a CO2 incubator. 7) Add 1ml of medium containing 2 times the normal serum and antibiotics concentration (2x medium) without removing the transfection mixture. If toxicity is a problem, remove the transfection mixture and replace with normal growth medium. 8) Incubate the cells an additional h. 9) Aspirate the medium and replace with fresh 1x medium containing the additives normally used to culture the cells. 10) Assay the cells using the appropriate protocol h after the addition of fresh medium in step 10 For the use of other transfection reagents, please follow the manufacturers' recommended protocol. MoleculA pdsipher technical manual January 2005
5 8 Selection of Plasmid Using Neomycin There are two main benefits for taking this approach: For difficult to transfect cell types, using neomycin selection will remove those cells that have not been transfected, leaving those cells that have successfully taken up the plasmid DNA. A stable cell line can be established, resulting in long-term shrna expression and target gene knockdown. To achieve this, the following protocol should be followed: 1) Perform a transient transfection as detailed above to assay the level of knockdown. 2) Following the transfection procedures detailed above, 24 hours post-transfection lift the cells from the plates using trypsin. Add G418 at a concentration of between 50 and 1500 µg/ml µg/ml is a good starting point. 3) Examine the wells every 48 hours to look for viable cells. Choose the lowest concentration of G418 that results in widespread cell death after 7 to 9 days, and kills all wild-type cells after 14 days. Use this concentration to select for resistant cell lines. Note: If your target gene is essential for cell viability, a stable cell line may not be obtained. 4) Linearizing the vector prior to transfection to aid integration into the host genome can increase the efficiency of the establishment of a stable cell line. Linearization should be performed with a restriction enzyme that does not cut in any of the vector's functional cassettes (e.g. The HI promoter or the GFP expression cassette) such as MluI. 9 Technical Support For technical assistance please contact us at info@molecula.com. MoleculA pdsipher technical manual January 2005
Product: Arrest-In TM Transfection Reagent for RNAi
Product: Arrest-In TM Transfection Reagent for RNAi Catalog #: ATR1740, ATR1741, ATR1742, ATR1743 Product Description Arrest-In transfection reagent is a proprietary polymeric formulation, developed and
More informationTransfection of CRISPR/Cas9 Nuclease NLS ribonucleoprotein (RNP) into adherent mammalian cells using Lipofectamine RNAiMAX
Transfection of CRISPR/Cas9 Nuclease NLS ribonucleoprotein (RNP) into adherent mammalian cells using Lipofectamine RNAiMAX INTRODUCTION The CRISPR/Cas genome editing system consists of a single guide RNA
More informationTransIT -LT1 Transfection Reagent
Quick Reference Protocol, MSDS and Certificate of Analysis available at mirusbio.com/2300 INTRODUCTION TransIT -LT1 Transfection Reagent is a broad spectrum reagent that provides high efficiency plasmid
More informationTOOLS sirna and mirna. User guide
TOOLS sirna and mirna User guide Introduction RNA interference (RNAi) is a powerful tool for suppression gene expression by causing the destruction of specific mrna molecules. Small Interfering RNAs (sirnas)
More informationjetprime in vitro DNA & sirna transfection reagent PROTOCOL
jetprime in vitro DNA & sirna transfection reagent PROTOCOL DESCRIPTION jetprime is a novel powerful transfection reagent based on a polymer formulation manufactured at Polyplus-transfection. jetprime
More informationTransIT-TKO Transfection Reagent
Quick Reference Protocol, MSDS and Certificate of Analysis available at mirusbio.com/2150 INTRODUCTION TransIT-TKO is a broad spectrum sirna transfection reagent that enables high efficiency sirna delivery
More informationAvalanche -Omni Transfection Reagent
The Transfection & Gene Expression Experts Avalanche -Omni Transfection Reagent Cat. No. EZT-OMNI-1 Description Size: 0.75 ml 1.5 ml Store at 4 C Avalanche -Omni Transfection Reagent is a proprietary lipid-polymer
More informationTransIT -Lenti Transfection Reagent
Quick Reference Protocol, SDS and Certificate of Analysis available at mirusbio.com/6600 INTRODUCTION Lentivirus is an enveloped, single-stranded RNA virus from the Retroviridae family capable of infecting
More informationConvoy TM Transfection Reagent
Convoy TM Transfection Reagent Catalog No.11103 0.25ml (40-80 transfections in 35mm dishes) Catalog No.11105 0.5 ml (80-165 transfections in 35mm dishes) Catalog No.11110 1.0 ml (165-330 transfections
More informationTransIT Transfection Reagent
INTRODUCTION TransIT -2020 is a broad spectrum transfection reagent that provides superior transfection of plasmid DNA into mammalian cells. TransIT-2020 is suitable for both transient and stable transfection
More informationTransIT -BrCa Transfection Reagent
Quick Reference Protocol, MSDS and Certificate of Analysis available at mirusbio.com/5500 INTRODUCTION TransIT -BrCa Transfection Reagent is specifically optimized to provide exceptional transfection efficiency
More informationTransIT -Keratinocyte Transfection Reagent
TransIT -Keratinocyte Transfection Reagent Quick Reference Protocol, MSDS and Certificate of Analysis available at mirusbio.com/2800 INTRODUCTION TransIT -Keratinocyte Transfection Reagent is specifically
More informationTransIT -293 Transfection Reagent
TransIT -293 Transfection Reagent Quick Reference Protocol, MSDS and Certificate of Analysis available at mirusbio.com/2700 INTRODUCTION TransIT -293 Transfection Reagent is specifically optimized to provide
More informationGenScript Vector-based sirna Protocol (CMV Promoter)
GenScript Vector-based sirna Protocol (CMV Promoter) Technical Manual No. 0172 Version 20051115 I Introduction. 1 II Vector-based sirna 1 III sirna Expression Vector Using CMV Promoter... 2 IV Product
More informationTHE DELIVERY EXPERTS PROTOCOL
THE DELIVERY EXPERTS jetprime in vitro DNA & sirna transfection reagent PROTOCOL DESCRIPTION jetprime is a novel powerful molecule based on a polymer formulation manufactured at Polyplustransfection. jetprime
More informationTransIT -CHO Transfection Kit
Quick Reference Protocol, MSDS and Certificate of Analysis available at mirusbio.com/2170 INTRODUCTION TransIT -CHO Transfection Kit is specifically optimized to provide exceptional transfection efficiency
More informationUser s Guide MegaTran 1.0 Transfection Reagent
User s Guide MegaTran 1.0 Transfection Reagent Package Contents and Storage Conditions... 2 Related products... 2 Introduction... 2 Production and Quality Assurance:... 3 Experimental Procedures... 3 Transfection
More informationXactEdit Cas9 Nuclease with NLS User Manual
XactEdit Cas9 Nuclease with NLS User Manual An RNA-guided recombinant endonuclease for efficient targeted DNA cleavage Catalog Numbers CE1000-50K, CE1000-50, CE1000-250, CE1001-250, CE1001-1000 Table of
More informationTransIT -mrna Transfection Kit
INTRODUCTION TransIT -mrna Transfection Kit is designed to transfect RNA into a broad range of cell types with minimal cellular toxicity. RNA delivery avoids transcriptional regulation effects by directly
More informationSureSilencing shrna Plasmid Handbook
March 2011 SureSilencing shrna Plasmid Handbook For genomewide, plasmid-based RNA interference Sample & Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative
More informationCat # Box1 Box2. DH5a Competent E. coli cells CCK-20 (20 rxns) 40 µl 40 µl 50 µl x 20 tubes. Choo-Choo Cloning TM Enzyme Mix
Molecular Cloning Laboratories User Manual Version 3.3 Product name: Choo-Choo Cloning Kits Cat #: CCK-10, CCK-20, CCK-096, CCK-384 Description: Choo-Choo Cloning is a highly efficient directional PCR
More informationTHE DELIVERY EXPERTS. INTERFERin in vitro sirna transfection reagent PROTOCOL
THE DELIVERY EXPERTS INTERFERin in vitro sirna transfection reagent PROTOCOL DESCRIPTION INTERFERin is a powerful sirna transfection reagent that ensures efficient gene silencing and reproducible transfection
More informationCignal Reporter Assay Handbook
January 2011 Cignal Reporter Assay Handbook For cell-based pathway activity assays Sample & Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and
More informationTransIT -LT1 Transfection Reagent
INTRODUCTION TransIT -LT1 Transfection Reagent is a broad spectrum reagent that provides high efficiency plasmid DNA delivery in many mammalian cell types including primary cells. TransIT-LT1 is a low
More informationTransIT Transfection Reagent
INTRODUCTION TransIT -2020 Transfection Reagent is a high-performance, animal-origin free, broad spectrum reagent that provides exceptional transfection of plasmid DNA into mammalian cells. TransIT-2020
More informationLecture Four. Molecular Approaches I: Nucleic Acids
Lecture Four. Molecular Approaches I: Nucleic Acids I. Recombinant DNA and Gene Cloning Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single
More informationINTERFERin sirna transfection reagent
INTERFERin in vitro sirna Transfection Protocol Company Information... 2 Product Information... 3 1. Standard sirna transfection of adherent cells... 4 (forward transfection)... 4 1.1 Cell seeding... 5
More informationTransIT -293 Transfection Reagent
INTRODUCTION TransIT -293 Transfection Reagent is specifically optimized to provide exceptional transfection efficiency of plasmid DNA in HEK 293 and cell types of associated lineage. TransIT-293 provides
More informationGuide-it Indel Identification Kit User Manual
Clontech Laboratories, Inc. Guide-it Indel Identification Kit User Manual Cat. No. 631444 (120114) Clontech Laboratories, Inc. A Takara Bio Company 1290 Terra Bella Avenue, Mountain View, CA 94043, USA
More informationphcmv Expression Vectors Instruction Manual
phcmv Expression Vectors Instruction Manual Catalog Numbers P003100 P003200 P003300 A Division of Gene Therapy Systems, Inc. 10190 Telesis Court San Diego, CA 92121 Phone: 888-428-0558 (US. Toll-Free)
More informationTransIT -LT1 Transfection Reagent
Protocol for Product Nos. MIR 2300, 2304, 2305, 2306 i n t r o d u c t i o n The easy to use TransIT-LT1 (Low Toxicity) Transfection Reagent provides superior transfection efficiency, cell viability, and
More informationPureSpin DNA Clean Up Kit
PureSpin DNA Clean Up Kit Micro Columns INSTRUCTION MANUAL KIT COMPONENTS For Research Use Only PureSpin DNA Clean Up Kit, Micro Columns w/out Caps (Kit Size) OD2080 (50 Preps.) OD2080-2 (200 Preps.) Storage
More informationTransfection Kit Protocol for MIR 2900, 2904, 2905, 2906
TransIT-HeLaMONSTER Transfection Kit INTRODUCTION TransIT-HeLaMONSTER Transfection Kit is specifically optimized to provide exceptional transfection efficiency of plasmid DNA in HeLa cells and cell types
More informationViaFect Transfection Reagent
TECHNICAL MANUAL ViaFect Transfection Reagent Instructions for Use of Products E4981, E4982 and E4983 Revised 7/17 TM409 ViaFect Transfection Reagent All technical literature is available at: www.promega.com/protocols/
More informationsirna Transfection Into Primary Neurons Using Fuse-It-siRNA
sirna Transfection Into Primary Neurons Using Fuse-It-siRNA This Application Note describes a protocol for sirna transfection into sensitive, primary cortical neurons using Fuse-It-siRNA. This innovative
More informationColor-Switch CRE recombinase stable cell line
Color-Switch CRE recombinase stable cell line Catalog Number Product Name / Description Amount SC018-Bsd CRE reporter cell line (Bsd): HEK293-loxP-GFP- RFP (Bsd). RFP" cassette with blasticidin antibiotic
More informationAlt-R CRISPR-Cpf1 System:
user guide Alt-R CRISPR-Cpf1 System: Delivery of ribonucleoprotein complexes in HEK-293 cells using the Amaxa Nucleofector System See what more we can do for you at www.idtdna.com. For Research Use Only
More informationsirna Transfection Reagent
Description RiboJuice 0.3 ml 71115-3 1.0 ml 71115-4 Description RiboJuice efficiently delivers small interfering RNA (sirna) into a wide range of mammalian cell lines for targeted gene suppression (1).
More informationINTERFERin in vitro sirna/mirna transfection reagent PROTOCOL
INTERFERin in vitro sirna/mirna transfection reagent PROTOCOL DESCRIPTION INTERFERin is a powerful sirna/mirna transfection reagent that ensures efficient gene silencing and reproducible transfection in
More informationSynthetic Biology for
Synthetic Biology for Plasmids and DNA Digestion Plasmids Plasmids are small DNA molecules that are separate from chromosomal DNA They are most commonly found as double stranded, circular DNA Typical plasmids
More informationsirna transfection optimization with the Agilent 2100 bioanalyzer Application Note A new method for effective gene silencing
sirna transfection optimization with the Agilent 2100 bioanalyzer A new method for effective gene silencing Application Note Abstract Marc Valer Carsten Buhlmann Ioanna Andreou Martin Weber When working
More informationTransIT -Prostate Transfection Kit
INTRODUCTION TransIT -Prostate Transfection Kit is specifically optimized to provide exceptional transfection efficiency of plasmid DNA in Prostate cell types. Generally, prostate cell types have been
More informationPrecisionX Multiplex grna Cloning Kit. Cat. # CAS9-GRNA-KIT. User Manual
PrecisionX Multiplex grna Cloning Kit Store at -20 C upon receipt A limited-use label license covers this product. By use of this product, you accept the terms and conditions outlined in the Licensing
More informationIP-Free Electra DAUGHTER Vectors Mammalian with CMV promoter
IP-Free Electra DAUGHTER Vectors Mammalian with CMV promoter Electra cloning DNA2.0 has developed a simple one-tube universal cloning process that can be performed in a 5 minute bench-top reaction with
More informationConversion of plasmids into Gateway compatible cloning
Conversion of plasmids into Gateway compatible cloning Rafael Martinez 14072011 Overview: 1. Select the right Gateway cassette (A, B or C). 2. Design primers to amplify the right Gateway cassette from
More informationTransfection Reagent INTRODUCTION SPECIFICATIONS MATERIALS. For Research Use Only. Materials Supplied. Materials required, but not supplied
TransIT-Neural Transfection Reagent INTRODUCTION TransIT-Neural Transfection Reagent is specifically optimized to provide exceptional transfection efficiency of plasmid DNA in various neural cell types.
More informationDesigning and creating your gene knockout Background The rada gene was identified as a gene, that when mutated, caused cells to become hypersensitive
Designing and creating your gene knockout Background The rada gene was identified as a gene, that when mutated, caused cells to become hypersensitive to ionizing radiation. However, why these mutants are
More informationGateway Cloning Protocol (Clough Lab Edition) This document is a modification of the Gateway cloning protocol developed by Manju in Chris Taylor's lab
Gateway Cloning Protocol (Clough Lab Edition) This document is a modification of the Gateway cloning protocol developed by Manju in Chris Taylor's lab With the Gateway cloning system, a PCR fragment is
More informationsirna Overview and Technical Tips
1 sirna Overview and Technical Tips 2 CONTENTS 3 4 5 7 8 10 11 13 14 18 19 20 21 Introduction Applications How Does It Work? Handy Tips Troubleshooting Conclusions Further References Contact Us 3 INTRODUCTION
More information#FD µl (for 200 rxns) Expiry Date: Description. 1 ml of 10X FastDigest Green Buffer. Store at -20 C
PRODUCT INFORMATION Thermo Scientific FastDigest SalI #FD0644 Lot: 5'...G T C G A C...3' 3'...C A G C T G...5' Supplied with: Store at -20 C 200 µl (for 200 rxns) Expiry Date: BSA included www.thermoscientific.com/onebio
More informationAlt-R CRISPR-Cas9 system:
Alt-R CRISPR-Cas9 system: Delivery of ribonucleoprotein complexes into Jurkat T cells using the Bio-Rad Gene Pulser Xcell Electroporation System See what more we can do for you at www.idtdna.com. For Research
More informationThe following manual contains information about and Instructions for the following psuper.retro vectors: VEC-PRT-0002 (circular)
psuper.retro.puro : Manual A Vector System for Expression of Short Interfering RNA The following manual contains information about and Instructions for the following psuper.retro vectors: Vector Name psuper.retro.puro
More informationTrueORF TM cdna Clones and PrecisionShuttle TM Vector System
TrueORF TM cdna Clones and PrecisionShuttle TM Vector System Application Guide Table of Contents Package Contents and Storage Conditions... 2 Related, Optional Reagents... 2 Related Products... 2 Available
More informationManipulation of Purified DNA
Manipulation of Purified DNA To produce the recombinant DNA molecule, the vector, as well as the DNA to be cloned, must be cut at specific points and then joined together in a controlled manner by DNA
More informationCELLTECHGEN For Research Only. Construction of all-in-one vector for Lenti-virus system (Example: Lenti-EF1 -Cas9-EGFP-U6 sgrna vector)
Construction of all-in-one vector for Lenti-virus system (Example: Lenti-EF1 -Cas9-EGFP-U6 sgrna vector) Catalog number: CTG-CAS9-18 Introduction The vector Lenti-EF1 -Cas9-EGFP-U6 sgrna is designed for
More informationAverage Yields* Yeast DNA Yeast RNA Time to Complete 10 Purifications * Yield will vary depending on the type of sample processed
3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com Fungi/Yeast RNA/DNA Purification Kit Product # 35800 Product Insert
More informationjetcrispr RNP transfection reagent PROTOCOL
jetcrispr RNP transfection reagent PROTOCOL DESCRIPTION jetcrispr is a RiboNucleoProtein (RNP) transfection reagent designed to perform CRISPR-Cas9 genome editing in mammalian cells. This reagent has been
More informationCHOgro Expression System
SDS and Certificate of Analysis available at mirusbio.com/6260 INTRODUCTION The CHOgro Expression System is an optimized platform for transient, high titer protein production in suspension CHO derived
More informationCELLTECHGEN For Research Only. Construction of sgrna expression vector for Lenti-virus system (Example: Lenti-U6 sgrna-ef1 -Puro vector)
Construction of sgrna expression vector for Lenti-virus system (Example: Lenti-U6 sgrna-ef1 -Puro vector) Catalog number: CTG-CAS9-11 Introduction The vector Lenti-U6 sgrna-ef1 -Puro is designed for expression
More information1. COMPONENTS. PyroStart Fast PCR Master Mix (2X) (#K0211 for 250 reactions of 20µl) 2. STORAGE 3. DESCRIPTION
1 2 1 PyroStart Fast PCR Master Mix (2X) (#K0211 for 250 reactions of 20µl) TABLE OF CONTENTS 1. COMPONENTS... 2 2. STORAGE... 2 3. DESCRIPTION... 2 4. PROTOCOL FOR FAST PCR... 3 4.1. General Considerations...
More informationDiagnosis Sanger. Interpreting and Troubleshooting Chromatograms. Volume 1: Help! No Data! GENEWIZ Technical Support
Diagnosis Sanger Interpreting and Troubleshooting Chromatograms GENEWIZ Technical Support DNAseq@genewiz.com Troubleshooting This troubleshooting guide is based on common issues seen from samples within
More informationProduct Name : Simple mirna Detection Kit
Product Name : Simple mirna Detection Kit Code No. : DS700 This product is for research use only Kit Contents This kit provides sufficient reagents to perform 20 reactions for detecting microrna. Components
More informationApplication Note sirna dependent gene silencing in HeLa cells cultivated on various cell culture surfaces
Application Note sirna dependent gene silencing in HeLa cells cultivated on various cell culture surfaces 1 Introduction 1.1 RNA interference (RNAi) One of the most important advances in biology in the
More informationCustom RNAi Services. GeneCust Europe. GeneCust Europe
GeneCust Europe Laboratoire de Biotechnologie du Luxembourg S.A. 2 route de Remich L-5690 Ellange Luxembourg Tél. : +352 27620411 Fax : +352 27620412 Email : info@genecust.com Web : www.genecust.com Custom
More informationThe RNAi Consortium (TRC) Broad Institute
TRC Laboratory Protocols Protocol Title: Lentivirus production of shrna, CRISPR, or ORF-pLX clones in 10 cm dishes or 6- well plates Current Revision Date: 6/3/2015 RNAi Platform,, trc_info@broadinstitute.org
More informationRNA Clean-Up and Concentration Kit Product # 23600, 43200
3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com RNA Clean-Up and Concentration Kit Product # 23600, 43200 Product
More informationjetpei -Macrophage in vitro DNA transfection reagent PROTOCOL
jetpei - in vitro DNA transfection reagent PROTOCOL DESCRIPTION jetpei - allows DNA transfection of macrophages and macrophage-like cells. It contains a mannose-conjugated linear polyethylenimine that
More informationSureSilencing sirna Array Technology Overview
SureSilencing sirna Array Technology Overview Pathway-Focused sirna-based RNA Interference Topics to be Covered Who is SuperArray? Brief Introduction to RNA Interference Challenges Facing RNA Interference
More informationSANTA CRUZ BIOTECHNOLOGY, INC.
TECHNICAL SERVICE GUIDE: Western Blotting 2. What size bands were expected and what size bands were detected? 3. Was the blot blank or was a dark background or non-specific bands seen? 4. Did this same
More informationAbsolute Human Telomere Length Quantification qpcr Assay Kit (AHTLQ) Catalog # reactions
Absolute Human Telomere Length Quantification qpcr Assay Kit (AHTLQ) Catalog #8918 100 reactions Product Description Telomeres are repetitive nucleotide elements at the ends of chromosomes that protect
More informationTransIT -Lenti Transfection Reagent
Quick Reference Protocol, SDS and Certificate of Analysis available at mirusbio.com/6600 INTRODUCTION Lentivirus is an enveloped, single-stranded RNA virus from the Retroviridae family capable of infecting
More informationTransIT-PRO Transfection Reagent Protocol for MIR 5740 and 5750
Quick Reference Protocol, SDS and Certificate of Analysis available at mirusbio.com/5740 INTRODUCTION TransIT-PRO Transfection Reagent was developed by empirically testing proprietary lipid and polymer
More informationNon-Organic-Based Isolation of Mammalian microrna using Norgen s microrna Purification Kit
Application Note 13 RNA Sample Preparation Non-Organic-Based Isolation of Mammalian microrna using Norgen s microrna Purification Kit B. Lam, PhD 1, P. Roberts, MSc 1 Y. Haj-Ahmad, M.Sc., Ph.D 1,2 1 Norgen
More informationQuant One Step RT-PCR Kit
1. Quant One Step RT-PCR Kit For fast and sensitive one-step RT-PCR www.tiangen.com/en RT121221 Quant One Step RT-PCR Kit Kit Contents Cat. no. KR113 Contents Hotmaster Taq Polymerase (2.5 U/μl) Quant
More informationMolecular Cell Biology - Problem Drill 11: Recombinant DNA
Molecular Cell Biology - Problem Drill 11: Recombinant DNA Question No. 1 of 10 1. Which of the following statements about the sources of DNA used for molecular cloning is correct? Question #1 (A) cdna
More information3 Designing Primers for Site-Directed Mutagenesis
3 Designing Primers for Site-Directed Mutagenesis 3.1 Learning Objectives During the next two labs you will learn the basics of site-directed mutagenesis: you will design primers for the mutants you designed
More informationConcepts and Methods in Developmental Biology
Biology 4361 Developmental Biology Concepts and Methods in Developmental Biology June 16, 2009 Conceptual and Methodological Tools Concepts Genomic equivalence Differential gene expression Differentiation/de-differentiation
More informationCells and Tissue DNA Isolation Kit (Magnetic Bead System) 50 Preps Product # 59100
3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com Cells and Tissue DNA Isolation Kit (Magnetic Bead System) 50 Preps
More informationPlasmid Midiprep Plus Purification Kit. Cat. # : DP01MD-P10/ DP01MD-P50 Size : 10/50 Reactions Store at RT For research use only
Plasmid Midiprep Plus Purification Kit Cat. # : DP01MD-P10/ DP01MD-P50 Size : 10/50 Reactions Store at RT For research use only 1 Description: The Plasmid Midiprep Plus Purification Kit provides simple
More informationjetpei -FluoF in vitro DNA Transfection Protocol
jetpei -FluoF in vitro DNA Transfection Protocol Company Information... 2 Product Information... 3 Contact our Technical Assistance and Scientific Advice Service... 4 1.Transfection protocol... 5 1. 1.
More informationKOD -Plus- Mutagenesis Kit
Instruction manual KOD -Plus- Mutagenesis Kit 0811 F0936K KOD -Plus- Mutagenesis Kit SMK-101 20 reactions Store at -20 C Contents [1] Introduction [2] Flow chart [3] Components [4] Notes [5] Protocol 1.
More informationKit Specifications 45 g. 45 g of RNA 8 L
RNA Clean-Up and Concentration Micro-Elute Kit Product # 61000 Product Insert Norgen s RNA Clean-Up and Concentration Micro-Elution Kit provides a rapid method for the purification, cleanup and concentration
More informationPhosphate buffered saline (PBS) for washing the cells TE buffer (nuclease-free) ph 7.5 for resuspending the SingleShot RNA control template
Catalog # Description 172-5085 SingleShot SYBR Green Kit, 100 x 50 µl reactions For research purposes only. Introduction The SingleShot SYBR Green Kit prepares genomic DNA (gdna) free RNA directly from
More informationpsirna-hh1neo G2 A simple and innovative tool to create sirnas Catalog # psirna2-n11g2c For research use only Version # 03F13-MT
psirna-hh1neo G2 A simple and innovative tool to create sirnas Catalog # psirna2-n11g2c For research use only Version # 03F13-MT PRODUCT INFORMATION Content: - 20 µg of lyophilized circular psirna-hh1neo
More informationRayBio Apoptotic DNA Ladder Extraction Kit
RayBio Apoptotic DNA Ladder Extraction Kit User Manual Version 1.1 March 1, 2016 RayBio Apoptotic DNA Ladder Extraction (Cat#: 68SO-DNAL-S50) RayBiotech, Inc. We Provide You With Excellent Support And
More informationOPPF-UK Standard Protocols: Mammalian Expression
OPPF-UK Standard Protocols: Mammalian Expression Joanne Nettleship joanne@strubi.ox.ac.uk Table of Contents 1. Materials... 3 2. Cell Maintenance... 4 3. 24-Well Transient Expression Screen... 5 4. DNA
More informationLentiviral shrna expression Cloning Kit User Manual for generating shrna expression lentivectors
Lentiviral shrna expression Cloning Kit User Manual for generating s Cat# Product Name Amount Application LTSH-H1-GB peco-lenti-h1-shrna(gfp-bsd) cloning kit with GFP-Blasticidin selection LTSH-H1-GP peco-lenti-h1-shrna(gfp-puro)
More informationFectoPRO DNA transfection kit for Bioproduction PROTOCOL
DNA transfection kit for Bioproduction PROTOCOL DESCRIPTION transfection kit is specifically designed for enhanced Transient Gene Expression using low DNA amounts, in suspension CHO and HEK-293 cells as
More informationYeaStar Genomic DNA Kit Catalog No. D2002
Instructions YeaStar Genomic DNA Kit Catalog No. D2002 Table of Contents General Information...... 1 Package Contents Ordering Information General Description...... 2 Protocol I.......... 2 Protocol II.
More informationE.Z.N.A. mirna Kit. R preps R preps R preps
E.Z.N.A. mirna Kit R7034-00 5 preps R7034-01 50 preps R7034-02 200 preps August 2011 E.Z.N.A. Micro RNA Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Preparing
More informationElectroporation of human induced pluripotent stem (ips) cells
Electroporation of human induced pluripotent stem (ips) cells Ribonucleoprotein delivery using the Alt-R CRISPR-Cas9 System and the NEPA21 Electroporator Contributed by Nepa Gene, Chiba, Japan (info@nepagene.jp)
More informationGeneCellin TM Transfection Reagent Protocol
GeneCellin TM Transfection Reagent Protocol Table of content Description......3 Content......3 Storage......3 Certificate of quality......4 Parameters influencing transfection efficiency......4 Nucleic
More informationHiPer RT-PCR Teaching Kit
HiPer RT-PCR Teaching Kit Product Code: HTBM024 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 4 hours Agarose Gel Electrophoresis: 45 minutes Storage Instructions: The
More informationSilencer Select Pre-designed sirna Silencer Select Validated sirna Silencer Select Custom Designed sirna Custom Select sirna
Catalog #: Various Silencer Select Pre-designed sirna Silencer Select Validated sirna Silencer Select Custom Designed sirna Custom Select sirna General Product Details and User Information Refer to page
More informationsherwood UltramiR shrna Lentiviral Target Gene Set in pzip
sherwood UltramiR shrna Lentiviral Target Gene Set in pzip TLHSU1400 (human), TLMSU1400 (mouse), TLRSU1400 (rat) Format: Bacterial glycerol stock sherwood-ultramir shrna sherwood-ultramir short hairpin
More informationDuraScribe T7 Transcription Kit
DuraScribe T7 Transcription Kit Cat. Nos. DS010910 and DS010925 Available exclusively thru Lucigen. lucigen.com/epibio www.lucigen.com MA170E DuraScribe T7 Transcription Kit 7/2017 1 1. Introduction The
More informationLaboratory #7 PCR PCR
1 Laboratory #7 Polymerase chain reaction () is DNA replication in a test tube. In vitro enzymatic amplification of a specific segment of DNA. Many Applications. direct cloning from DNA or cdna. Mutagenesis
More informationBACMAX DNA Purification Kit
Cat. No. BMAX044 Connect with Epicentre on our blog (epicentral.blogspot.com), Facebook (facebook.com/epicentrebio), and Twitter (@EpicentreBio). www.epicentre.com Lit. # 212 10/2012 1 EPILIT212 Rev. A
More informationFosmidMAX DNA Purification Kit
Cat. No. FMAX046 Connect with Epicentre on our blog (epicentral.blogspot.com), Facebook (facebook.com/epicentrebio), and Twitter (@EpicentreBio). www.epicentre.com Lit. # 204 10/2012 1 EPILIT204 Rev. A
More informationChromatrap 96 DNA Micro Elution Purification Kit
V 1.0 Chromatrap 96 DNA Micro Elution Purification Kit High throughput plates for ultra-pure DNA Protocol v1.0 Catalogue no. 500240 ADVANCEMENTS IN EPIGENETICS Contents Kit Components and Storage 3 Additional
More information