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We narrowed to 18 results for: total dna

Showing: 11 - 18 of 18 results

  1. Protocol - How to Perform a Diagnostic Digest

    Type
    Protocol
    ...You may also like... Restriction Digest of Plasmid DNA Agarose Gel Electrophoresis Introduction Restriction...bacterial endonucleases that recognize a large range of DNA sequences. Given the variety of these enzymes and...scientists employ to selectively move a specific piece of DNA from one plasmid to another. A diagnostic restriction...advantage of the fact that restriction enzymes cleave DNA at specific sequences called restrictions sites. ...restriction enzyme or enzymes that cut your plasmid. Many DNA analysis tools, including Addgene’s Sequence Analyzer...Electrophoresis chamber Pipetman Pipet tips Reagents Liquid DNA aliquot of your plasmid of interest (see below for...Gel loading dye Electrophoresis buffer Verifying Total Plasmid Size -OR- Insert and Backbone Size The simplest...

  2. Plasmid Modification by Annealed Oligo Cloning (with Protocols)

    Type
    Protocol
    ...may also like... Restriction Digest of Plasmid DNA DNA Ligation Bacterial Transformation Summary Oligo...cut out the band containing your vector DNA. Gel purify your DNA away from the agarose using a commercially... used anytime you need to add a short stretch of DNA to a plasmid, such as: Changing a Multiple Cloning...concentrations. We recommend mixing 2μg each in a total volume of 50μL - add additional annealing buffer...

  3. Kit Free RNA Extraction

    Type
    Protocol
    ...Extraction Without a Kit You may also like... Kit-Free DNA Purification Agarose Gel Purification Molecular Biology...extraction. Before Starting RNA is not as stable as DNA and is susceptible to degradation by heat, RNases...Option B): LiCl selectively precipitates RNA versus DNA or proteins. Add the correct amount of 7.5 M LiCl...supernatant. There should be a gel-like white pellet of total RNA in the bottom of the tube. Wash the RNA by resuspending...nucleic acid quantification, see our protocol for DNA quantification , which can be modified for RNA. Store...

  4. Lentivirus ddPCR Titration

    Type
    Protocol
    ...aspirate supernatant. Extract genomic DNA according to the GeneJet Genomic DNA Purification Kit instructions....Thermo Scientific, 10199-452 Reagents GeneJet Genomic DNA Purification Kit, Thermo Fisher, K0721 6-well tissue...Dilution Factor Virus Volume to Dilute Media Volume Total Volume 2.5 160 µL viral stock 240 µL 400 µL 5 200...µL 900 nM, 250 nM Nuclease-free water 4 µL 36 µL Total Volume 16 µL 144 µL Vortex the master mix for 15...well is 300,000 and the virus volume refers to the total volume the cells were transduced, which is 1.5 mL...diluted and used to transduce HEK293T cells. Genomic DNA is extracted from the target cells and assayed for...

  5. General Transfection

    Type
    Protocol
    ...plasmid DNA purification should include an endotoxin removal step. For high quality plasmid DNA, the plasmid...to the diluted DNA. Add the diluted PEI dropwise while gently flicking the diluted DNA tube. Incubate ...strain such as NEB stable. Dilute 1:3 (µg DNA:µg PEI) in 500 µL total of OptiPro SFM (per 10 cm plate). 56.7...possible range of ratios to test: Ratio of DNA:PEI Amount of DNA (μg) Volume of 1 mg/mL PEI (μL) 1:1 18.9...25 mM chloroquine diphosphate. Dilute 18.9 µg of DNA into 500 µL of Opti-Pro SFM. Pro-Tip Endotoxins can...passage 20 for viral production. The optimal mass DNA:mass PEI ratio will need to be empirically determined...needs to be validated and the best ratio of mass DNA:mass PEI determined. Procedure Seed HEK293T cells at...

  6. Transfection for Recombinant Antibodies

    Type
    Protocol
    ...Reagents HEK293 cells Recombinant antibody plasmid DNA 500 mL vented flask 50 mL conical tubes Microcentrifuge..., Millipore Sigma A6279 Before Starting Warm the DNA and working stock of PEI-MAX to room temperature ...Transfer the PEI-MAX and recombinant antibody plasmid DNA sample to the biosafety cabinet and incubate for ...cabinet. Add 180 µg of recombinant antibody plasmid DNA to one tube of 6 mL BCD TFX. Cap the tube and vortex...1 to 1:6. Add the diluted PEI-MAX to the diluted DNA. Cap the tube and vortex with three 1 s pulses. Incubate...cells to the biosafety cabinet. Add 12 mL of PEI-MAX/DNA mix to the flask dropwise. Cap the flasks and swirl...Pro-Tip The feed can be repeated up to 4 times for a total of 16% of the culture volume. At 72-96 h post-transfection...

  7. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...measure the DNA concentration, use 1 μL) 2 μL 10x NEB T4 DNA ligase buffer 1 μL NEB T4 DNA ligase add ...#B7001S NEB buffer 2 NEB #B7002S T4 DNA ligase NEB #M0202S T4 DNA ligase buffer NEB #B0202S DH5 alpha ...will increase the chance of damaging the DNA. Purify the DNA using a Qiaquick gel extraction kit. Elute...kit to obtain DNA. 3. Conduct a restriction digest with EcoRI and NcoI: 1 μg miniprep DNA 2 μL 10x NEB ...transfect plasmid DNA directly into the target cells. The shRNA will be expressed, but the DNA is unlikely ... pLKO.1 TRC-cloning vector (maxiprep or miniprep DNA) 5 μL 10x NEB buffer 1 1 μL AgeI add ddH 2 O to bring...ddH 2 O Incubate at 37°C for 2 hours. Run digested DNA on 0.8% low melting point agarose gel until you can...

  8. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...purified AAV samples with DNase I to eliminate any contaminating plasmid DNA carried over from the production...add carrier DNA to a final concentration of 4 ug/mL to each standard dilution. Dilute DNase-treated and...nuclease free water (uL) Dilution factor Total dilution Dilution 1 (DNase step) 5 uL AAV stock 45 uL 10X 10X...Universal" SYBR master mix which contains a high-quality DNA polymerase and a blend of dTTP/dUTP to minimize carryover...ITR-containing plasmid for standard curve RNase-free DNase 10X DNase buffer Nuclease-free water Microcentrifuge ...production process (DNase does not penetrate the virion). 5 μL sample + 39 μL H 2 O + 5 μL 10X DNase buffer + 1... 1 μL DNase Gently mix sample (do not vortex) Incubate 30 min at 37 °C Transfer to ice ** Critical: do...
Showing: 11 - 18 of 18 results