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We narrowed to 1,035 results for: LAS

Showing: 821 - 840 of 1035 results

  1. Lentivirus Production

    Type
    Protocol
    ...DNAs: Lentiviral Packaging plasmid(s), Envelope plasmid, and Transfer plasmid encoding your insert of interest...cells in a T75 flask in 15 mL DMEM Complete. Wednesday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM...transfection plasmids into OptiPro SFM: Reagent Amount per 10 cm dish* Packaging Plasmid ( psPAX2 ) 1.3...1.3 pmol Envelope Plasmid ( pMD2.G ) 0.72 pmol Transfer Plasmid* 1.64 pmol OptiPro SFM to total volume...volume 500 μL *Plasmid concentrations and ratios should be optimized for each transfer plasmid. Pro-Tip Endotoxins...therefore, plasmid DNA purification should include an endotoxin removal step. For high quality plasmid DNA, ... cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA 27.8 μg), this would be...

  2. Protocol - Bacterial Transformation

    Type
    Protocol
    ... also like... Plasmid Cloning by Restriction Enzyme Digest Restriction Digest of Plasmid DNA DNA Ligation...both storing and replicating plasmids. Because of this, nearly all plasmids (even those designed for mammalian...that will help to maintain the plasmid without rearrangement of the plasmid DNA. Additionally, specific ...tube of plasmid DNA and just need to transform bacteria so that you can grow up more of the plasmid) you ...efficient at taking up larger plasmids. If you need to transform large plasmids, it is a good idea to use ...Learn how to transform E. coli with your plasmid of interest. Protocols...introduced into a cell. Transformation of bacteria with plasmids is important not only for studies in bacteria ...

  3. CRISPR Library Amplification

    Type
    Protocol
    ...differences, and plasmid recombination can all impact the representation of individual plasmids in the pooled... ). Lentiviral plasmids can recombine between their LTRs resulting in a smaller plasmid containing only...protocol to perform amplification of CRISPR pooled plasmid libraries in Escherichia coli cells. Protocols... protocol allows the amplification of a pooled-plasmid library in Escherichia coli cells. Pooled libraries...libraries contain tens to millions of different plasmids in a single sample. They are often used for screening...throughput multiplexed experiments. These pooled plasmids differ by only a short region of DNA: guide RNAs...results for libraries up to 200,000 individual plasmids and can be scaled according to the actual number...

  4. Transfection for Recombinant Antibodies

    Type
    Protocol
    ... HEK293 cells Recombinant antibody plasmid DNA 500 mL vented flask 50 mL conical tubes Microcentrifuge...Transfer the flask of HEK293 cells to the biosafety cabinet. Add 12 mL of PEI-MAX/DNA mix to the flask dropwise...dropwise. Cap the flasks and swirl 5–10 times to mix. Return the flask to the incubator. Section 3: BCD Feed...Recombinant Antibodies You may also like... Antibody Plasmid Collection Antibodies Blog Posts Molecular Biology...suspension HEK293 cells with recombinant antibody plasmids using Polyethylenimine Max as a transfection reagent...General Considerations For antibody-expressing plasmids containing the SV40 origin of replication use ... SV40 large T antigen. For antibody-expressing plasmids containing the Epstein-Barr virus origin of replication...

  5. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...μg pLKO.1 shRNA plasmid 750 ng psPAX2 packaging plasmid 250 ng pMD2.G envelope plasmid to 20 μl serum-...to vary the ratio of shRNA plasmid, packaging plasmid, and envelope plasmid to obtain the ratio that gives...shRNA. psPAX2 Packaging plasmid for producing viral particles. pMD2.G Envelope plasmid for producing viral... also be used with packaging plasmid pCMV-dR8.2 dvpr and envelope plasmid pCMV-VSVG from Robert Weinberg... should be worn at all times. Use plastic pipettes in place of glass pipettes or needles. Liquid waste....1 Protocol pLKO.1 - TRC Cloning Vector Addgene Plasmid 10878. Protocol Version 1.0. December 2006. Copyright... RNAi Consortium A.2 Map of pLKO.1 A.3 Related plasmids B. Designing shRNA Oligos for pLKO.1 B.1 Determine...

  6. AAV Production in HEK293 Cells

    Type
    Protocol
    ...of each plasmid needed to have a 1:1:1 molar ratio with 2 mg total DNA per CS5 Plasmid Plasmid Size (bp...conditions must be optimized. Plasmids for transfection: pHelper; pRC (Rep-Cap), plasmid expressing your gene ...cells from 2 x T-175 flasks. Cells should be at ~80% confluence. For each T-175 flask: Aspirate culture ...molar ratio of each plasmid: 2000 μg / 24,961 bp = 0.08 μg/bp Therefore, for each plasmid we need: Sample ...,180 cm 2 - the same surface area as 21 x T-175 flasks). Cell stacks provide an efficient means to scale-up...without having to handle a large number of T-175 flasks. Workflow Timeline Day 0: Seed cells in CS2 Day...Transfect cells Day 7 (am): Harvest cells Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL ...

  7. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...the assay multiple plasmids containing ITR were tested. Plasmid #59462 is one plasmid that gave reliable... calculated using this plasmid, but will change if you use a different plasmid. Sample Calculation Size...Size in bp of Addgene plasmid #59462 : 6208 bp Concentration of Addgene plasmid #59462 : 1.07 μg/μL Molecular...consistent results. Use the recommended plasmid, or test multiple plasmids to find a suitable one. Some labs... for a 96-well plate with 20 μL reaction volume. Last Update: February 13, 2019 Estimate of time required...ITR primer, 5'-CGGCCTCAGTGAGCGA ITR-containing plasmid for standard curve RNase-free DNase 10X DNase buffer...Nuclease Free Water 4.7 μL 470 μL Procedure Prepare a plasmid stock of 2 x 10 9 molecules/μL to generate a standard...

  8. Protocol - How to Create a Bacterial Glycerol Stock

    Type
    Protocol
    ...stocks of their plasmids. This way, when you want to make more plasmid DNA, the plasmid will already be... Storage of Plasmids Creating Bacterial Glycerol Stocks for Long-term Storage of Plasmids You may also...important for long-term storage of plasmids. Although you can store your plasmid DNA at -20°C, many labs also...also like... Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture Background Information... and growth temperature, can be found on your plasmid's information page. The next day you will be able...able to start an overnight culture for plasmid DNA prep the following day. Tips and FAQ The optimal concentration... glycerol stock the same time you prepare your plasmid DNA. In the morning, when you retrieve your liquid...

  9. Protocol - How to Perform Sequence Analysis

    Type
    Protocol
    ...flank the regions of your plasmid that you wish to confirm. Addgene's plasmid information pages recommend...Sequences" link on the Plasmid Information Pages. Addgene sequences the plasmid to verify tags, mutations...the entire plasmid. Addgene strongly recommends that you sequence any portion of the plasmid that is important...sequence analysis of a plasmid Protocols... Protocols Sequence Analysis Sequence Analysis of a Plasmid You may also like...Introduction Sequence verification of important plasmid features (such as the gene/insert, fusion proteins...common primers that are often found within the plasmid backbone (See Addgene's Sequencing Primers for ...

  10. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    ...transduced with lentiCas9-Blast and then selected with 1 µg/mL blasticidin for 9 days. Single cells ...cells for 13 days. lentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid #52962 ) and is described ...mammalian antibiotic resistance (e.g., puromycin, blasticidin), which enables selection of a stable cell culture... Expand and harvest stable cell lines Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL ...Appropriate antibiotic for selection (e.g. puromycin, blasticidin) Reagent Preparation DMEM Complete: 10% v/v FBS...obtaining accurate results. Check the cells for mycoplasma (Link opens in a new window) regularly Do not...stable cell pool, the antibiotic selection should last at least as long as it takes the control (untransduced...

  11. General Transfection

    Type
    Protocol
    ...cells in a 75 cm 2 flask in a volume of 15 mL. Wednesday: Plate 1x10 6 cells in a T75 flask in a volume of...therefore, plasmid DNA purification should include an endotoxin removal step. For high quality plasmid DNA, ...protocol to transfect mammalian cells with your plasmid of interest. Protocols...harvest cells, or perform your experiment Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL ...of 15 mL. Friday: Plate 8x10 5 cells in a T75 flask in a volume of 15 mL. Do not add antibiotics to the..., the plasmid should also be propagated in an endonuclease negative E. coli strain such as NEB stable....is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1–2 ...

  12. Protocol - How to Streak a Plate

    Type
    Protocol
    ...Agar Plates Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture Introduction If you ...or stab culture of bacteria and need to purify plasmid DNA from it, you will want to isolate an individual...will minimize the chance of having a mixture of plasmids in your purified DNA. This protocol explains how...bacterial colony by streaking it onto an LB agar plate. Last Update: Feb. 28, 2017 Protocol Video Watch the protocol...antibiotic. Label the bottom of the plate with the plasmid name and the date. It is also a good idea to add...through streak #2 and spread the bacteria over the last section of the plate, to create streak #3. Incubate...overnight (12-18 hours) at 37 °C. Pro-Tip Some plasmids or bacteria need to be grown at 30 °C instead ...

  13. Pouring LB Agar Plates

    Type
    Protocol
    ...also like... Plasmids 101 Blog Posts Mol Bio Protocols Cloning Protocols Introduction Plasmids can carry ...resistance gene on a plasmid allows researchers to easily isolate bacteria containing that plasmid from bacteria...resistance to that antibiotic - usually conferred by a plasmid carrying the antibiotic resistance gene. The following...LB/agar plates with your antibiotic of interest. Last Upload: September 14, 2016 Protocol Video Before...which can hold 10 - 15 mL of agar. Autoclavable flasks Sterile pipettes Ice bucket to hold antibiotic ...effective at selecting only bacteria containing the plasmids of interest (for example, fewer satellite colonies...at the right temperature, we recommend using a laser thermometer. Light the flame at the plate pouring...

  14. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ...transduced with lentiCas9-Blast 1 and then selected with 1 μg/mL blasticidin for 9 days. Single cells ...lentiCas9-Blast 1 . A549 cells were transduced (MOI = 37) and selected with 1 μg/mL blasticidin for 9 days...loading control. 1 lentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid #52962 ) and is described ...and expand monoclonal lines of interest Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL ...then expanded for an additional 3 weeks under blasticidin selection. Anti-Cas9 Western blotting was performed...2014 Aug;11(8):783-4. (Link opens in a new window) Last reviewed on: September 9, 2023...

  15. Gibson Assembly Protocol

    Type
    Protocol
    ...Design your plasmid and order primers (see figure to the right). When designing your plasmid, think about...correct plasmid product by restriction digest . Sequence the important regions of your final plasmid, particularly...efficient and effective method for the assembly of plasmids, and molecular biologists now use this method ...segments you will need to join to create your final plasmid. Adjacent segments should have identical sequences... background and enrich for correctly assembled plasmids by designing primers to split an antibiotic resistance...reaction if you're only making a small change in a plasmid (such as point mutations). Generate DNA segments...

  16. Western Blot

    Type
    Protocol
    ...follows: Remove the gel from the plastic packaging. Remove tape and plastic comb. Rinse the wells with deionized...Blot Western Blot You may also like... Antibody Plasmid Collection Antibody Blog Posts Antibody Guide Immunocytochemistry...adapted for wet and semi-dry transfer approaches. Last Update: January 24, 2022 Workflow Timeline Day 1...well microtiter plate Chemiluminescence substrate Plastic wrap Primary antibody Secondary antibody Deionized... white separator. Keep the Bottom Stack in the plastic tray. Place the Bottom Stack on the blotting surface...for 5 min at RT . Cover the membrane with clear plastic and use a gel imager with chemiluminescence detection...

  17. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ...Agar Plates Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture Introduction This protocol... plate and select transformed cells containing plasmids differing in their resistance genes, as one does...curve to determine optimal antibiotic concentration. Last Update: Oct. 27, 2017 Protocol Video Watch the protocol...or other antibiotic) E. coli transformed with a plasmid containing the carbenicillin (amp) resistance gene...effective at selecting only bacteria containing the plasmids of interest (for example, fewer satellite colonies...transform DH5α E. coli cells by heatshock with the plasmid of interest. See our transformation page for a ...

  18. Video Library

    Type
    Protocol
    ...Storing and Handling Addgene Plasmids What to do after you receive your plasmids from Addgene Instructions...Protocol Diagnostic Restriction Digest Verify a plasmid using restriction enzymes Diagnostic Restriction...Instructions for Storing and Handling Plasmids Genomic Deletions with CRISPR Generate genomic deletions in mammalian...Transformations We walk you through a standard plasmid transformation protocol and offer some tips and...Center How to Deposit Your guide to depositing plasmids with Addgene Depositing Page Career Videos (Link...

  19. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...Electrophoresis You may also like... Restriction Digest of Plasmid DNA Purifying DNA from an Agarose Gel DNA Ligation...a collection of DNA fragments of known lengths). Last Update: Feb. 20, 2018 Protocol Video Watch the protocol...agarose powder with 100 mL 1xTAE in a microwavable flask. See TAE Recipe . Pro-Tip TBE can be used instead...people prefer to microwave in pulses, swirling the flask occasionally as the solution heats up.). Caution...boil over. Placing saran wrap over the top of the flask can help with this, but is not necessary if you ... when you can comfortably keep your hand on the flask), about 5 mins. Optional : Add ethidium bromide ...

  20. Lab Safety for Biosafety Levels One and Two

    Type
    Protocol
    ...station if unwanted or biohazardous materials are splashed onto your face and/or get into your eyes. Wash...your skin, including, but not limited to broken glass or needles. Sharps containers must be thick walled...shaking or mixing, which may produce aerosols or splashes. These procedures should be done underneath a ...Working in a BSL-2 laboratory requires laboratory glasses in addition to coat and gloves. This lab coat should...here has been designed by the Addgene nonprofit plasmid repository and is being shared outside of Addgene...
Showing: 821 - 840 of 1035 results