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We narrowed to 45 results for: Ran

Showing: 11 - 20 of 45 results

  1. Fluorescence Titering Assay

    Type
    Protocol
    ...Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands and lots... were transduced with a range of dilutions of pHAGE-TO-dCas9-3XmCherry . 72 h post transduction, cells...cells transduced F = Fraction of cells with fluorescence D = Dilution Factor V T = Transduction Volume...Workflow Timeline Day 0: Seed 293T cells Day 1: Transduce cells Day 2 (am): Remove media, replace with fresh...to the media, as it can affect viability and transduction efficiency. Titer will vary between cell lines...1468.5 1.5 1:75 20 1478.5 1.5 1:100 15 1483.5 1.5 Untransduced 0 1498.5 1.5 Mix the dilutions well Gently aspirate... one dilution with one well remaining as the untransduced control). Count the cells in the remaining well...

  2. Lentivirus ddPCR Titration

    Type
    Protocol
    ... is based on an LV titer range of 1E+05–1E+09 TU/mL, where TU is transducing units and generally represents...July 7, 2023 Workflow Timeline Day 1: Seed and transduce cells Day 4: Treat cells with Benzonase and harvest...Complete 3 µL of 250 U/µL benzonase Procedure Transducing Cells Thaw the virus on ice. Prepare the following... pipette 10–20 times. Use a new pipette tip to transfer 200 µL of the first dilution into the next dilution... plate (1 dilution per well). Leave one well untransduced (add 150 µL of DMEM complete instead of a viral... into the wells containing the virus and the untransduced control. Pro-Tip For even seeding, prepare a...min. Resuspend cells in 500 µL DMEM complete and transfer to a microcentrifuge tube. Centrifuge for 5 min...

  3. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...step heats the double stranded DNA template strand to the point where the strands start denaturing and ...this temperature range to anneal to each of the single stranded DNA template strands. The DNA polymerase...of the sense and anti-sense strands of the template DNA. They are strands of nucleic acid that are starting...molar ratio. It also requires two unique single stranded DNA oligonucleotide (oligo) primers, which anneal...seconds at 94°C: Continued denaturation of double stranded DNA. Anneal primers for 30 seconds at 55°C: The...polymerase to synthesize and elongate the new target DNA strand accurately and rapidly. Repeat steps 2-4 25-30 ...fill-in any protruding ends of the newly synthesized strands. Materials List Reagents for each 50µL PCR: Thin-walled...

  4. CRISPR Library Amplification

    Type
    Protocol
    ... Day 1: Transform, recover, set up overnight growth (Estimated time 2-3 hours) Transformation should be...of 5 mL (3 mL SOC + 2 mL transformed Endura from two separate transformations). Pro-Tip Do not pipette...assumes familiarity with standard bacterial transformation and basic knowledge of the library being amplified...amplified. Please see our protocol on Bacterial Transformation . The generalized pooled library amplification...premixed LB (VWR 101414-072) for convenience but any brand or house- made LB that supports normal growth is...using a new vented falcon tube for every two transformations. Each of the four 14 mL Vented Falcon Tubes...repeatedly or mix when removing SOC containing transformed DNA-Endura from cuvette. Shake four 14 mL Vented...

  5. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ...incubation, transform DH5α E. coli cells by heatshock with the plasmid of interest. See our transformation page...for a detailed heatshock transformation protocol. Plate 50 µL of transformed E. coli /rescue media suspension...also like... Making LB Agar Plates Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture...plating antibiotic over-agar for the selection of transformed E. coli . Over-agar spreading of antibiotic makes...investigator to conveniently plate and select transformed cells containing plasmids differing in their ... sterile water (or other antibiotic) E. coli transformed with a plasmid containing the carbenicillin (...carbenicillin, plated over-agar for selection of transformed E. coli . Sample Data Selection of E.coli on ...

  6. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ...recipient plasmid backbone. Transformation Proceed with the transformation according to the manufacturer...from your transformation will give you the first indication as to whether your transformation worked. Your...Digest of Plasmid DNA DNA Ligation Bacterial Transformation Summary PCR based cloning is incredibly versatile...chance for mis-priming, so you can use a pretty wide range of annealing temperatures, but you may need to increase...competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent...conduct a positive control to ensure that your transformation worked. You could also try varying the amount...cloning. DNA replication by PCR has error rates that range from roughly 1 per 500bp to roughly 1 per 10 million...

  7. Plasmid Cloning by Restriction Enzyme Digest (with Protocols)

    Type
    Protocol
    ...self-ligating recipient plasmid backbone. Transformation Transform your ligation reaction into your bacterial...from your transformation will give you the first indication as to whether your transformation worked. our...Digest of Plasmid DNA DNA Ligation Bacterial Transformation Summary The following technique can be used...competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent...conduct a positive control to ensure that your transformation worked. You should also verify that you are...

  8. Ligation Independent Cloning

    Type
    Protocol
    ...now ready for transformation. Use 1-2 μl of annealing reaction for each transformation, following a standard...stability to hold the plasmid together through the transformation/replication process. LIC employs long overhangs...association between fragments, allowing for transformation without ligation. Because of its dual polymerase...same principle (homologous to the first G on 3'-5' strand from cut site). For simplicity, only the 5' primer...heating to 75° for 20 minutes. Step 6: Anneal and Transform Mix your treated vector and insert at a molar ...

  9. Video Library

    Type
    Protocol
    ... tips Bacterial Transformations We walk you through a standard plasmid transformation protocol and offer...tricks to reduce transformation time and increase efficiency Bacterial Transformation Protocol Inoculating...when requesting samples from Addgene Technology Transfer How to Order A step-by-step walkthrough of Addgene's...Addgene's Business Development Manager, discusses his transition from the Scientist Team to Business Team, his...

  10. Protocols for Molecular Biology, Plasmid Cloning, and Viral Preps

    Type
    Protocol
    ...practical lab protocols that you can use for a wide range of applications, with videos for select protocols...Ligation Assemble DNA using DNA ligase Bacterial Transformation Introduce DNA into a bacterial strain Watch...Virus Name Description Link to Video General Transfection Introduce plasmid DNA to mammalian cells Lentivirus...Produce lentivirus with a polyethyenimine (PEI) transfection protocol Fluorescence Titering Assay for Lentivirus...preparation Antibodies Name Description Link to Video Transfection Introduce plasmid DNA to mammalian cells to ...
Showing: 11 - 20 of 45 results