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We narrowed to 22 results for: abo.4

Showing: 1 - 20 of 22 results

  1. Affinity Purification of Recombinant Antibodies with Protein A or Protein G

    Type
    Protocol
    ...2 PO 4 ∙H 2 O) 610 mL of sterile sodium phosphate dibasic (NaH 2 PO 4 ) Store up to 1 month at 4 °C Adjust...phosphate monobasic monohydrate (NaH 2 PO 4 ∙H 2 O), pH 7.0 138 g NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust...sodium phosphate dibasic (NaH 2 PO 4 ), pH 7.0 142 g of NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust pH...up to 4 mL of recombinant antibody sample. Pro-Tip If the volume of the sample is greater than 4 mL , ...Equipment Class II, Type A2 Biological Safety Cabinet 4 °C Refrigerator Pipette controller Benchtop centrifuge...Protein A/G binding buffer and store in 20% ethanol at 4 °C . Pro-Tip Columns may be re-used up to 5x when ...Apply 15 mL of PBS to the reservoir of a Amicon Ultra-4 30 kDa MWCO column. Incubate for 10 min at RT . Centrifuge...

  2. AAV Production in HEK293 Cells

    Type
    Protocol
    ...needed. Add stir bar and stir slowly at 4 °C for 1 h, then keep at 4 °C for 3 h without stirring to allow...Reagent Preparation DMEM Complete : 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or stable alternative, such...glutaGRO 11 mL of 200 mM L-alanyl-L-glutamine. Store at 4 °C. D1 + 0.1 M sorbitol (optional) : DMEM low glucose... °C. After thawing, the solution can be stored at 4 °C for up to 2 months. After 2 months, discard the...solution may separate into phases. Aliquot and store at 4 °C. Cell Lysis Buffer : 50 mM Tris HCl, 150 mM NaCl... Tris HCl pH 8.5 + 60 mL of 5 M Sodium Chloride + 4 mL of 1 M Magnesium Chloride Close the bottle and ...Filter sterilize through a 0.22 μm membrane. Store at 4 °C. Considerations Before You Start The health of ...

  3. Kit Free RNA Extraction

    Type
    Protocol
    ...have a non-refrigerated microcentrifuge, see step 4 of either protocol before you start. Homogenizer Vortexer...freezer Reagents Solution D (for Protocol Option #1 ): 4 M Guanidinium Thiocyanate 25 mM Sodium Citrate, pH... #2) Water-saturated Phenol 2 M Sodium Acetate pH 4 Chloroform/Isoamyl alcohol (49:1) 75% Ethanol RNase-free...solution RNase free tubes: microcentrifuge tubes, 4 mL polypropylene tubes RNase decontamination solution...homogenized sample(s).Transfer tissue/cell lysate to a 4 mL tube. Add the following sequentially to 1 mL of...centrifuge the sample(s) for 15 minutes at 12,000 × g at 4°C to separate RNA from the rest of the tissue/cell...cell lysate. Pro-Tip Having your samples spin at 4°C helps reduce RNA degradation. If you don’t have access...

  4. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...days Protein knockdown (western blot) ≥4 days Phenotypic assay ≥4 days Back to Top G. Safety BL2 safety...solution is stable at 4°C for up to one year. The powder form of polybrene is stable at 4°C for several years... oligos C.3 Digesting pLKO.1 TRC-Cloning Vector C.4 Ligating and transforming into bacteria D. Screening...15-19. Avoid targeting introns. Avoid stretches of 4 or more nucleotide repeats, especially repeated Ts... 5 μL 10x NEB buffer 2 35 μL ddH 2 O Incubate for 4 minutes at 95°C in a PCR machine or in a beaker of...30 μL of ddH 2 O. Measure the DNA concentration. C.4 Ligating and Transforming into Bacteria Use your ligation... bring to 20 μL final volume Incubate at 16°C for 4-20 hours. Transform 2 μL of ligation mix into 25 μL...

  5. Immunocytochemistry

    Type
    Protocol
    ...coverslips HeLa cells 24-well plate 4% Paraformaldehyde 5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) Bovine...with 500 µL of cold 4% paraformaldehyde in PBS on ice for 15 min . Pro-Tip While 4% Paraformaldehyde fixation...20, 2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells Equipment Pipette controller ...water Microscope slide Anti-fade mounting medium Laboratory wipes 15 mL conical tubes 50 mL conical tubes...paraformaldehyde and follow your institution's laboratory safety guidelines for disposing of waste in the...remove the coverslip. Blot the coverslip with a laboratory wipe to remove excess liquid. Add 1 drop of anti-fade...

  6. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ...for 2 h at room temperature or overnight at 4 °C . Section 4: Secondary antibody incubation Carefully remove... over several days by incubating certain steps at 4 °C instead of room temperature or 37 °C. The protocol...: Blocking Day 3: Primary antibody incubation Day 4: Secondary antibody incubation and plate read Tips...and incubate at 37 °C for 30 min , or overnight at 4 °C . Section 2: Block the plate Prepare the wash buffer...400 rpm for 2 h at room temperature or overnight at 4 °C . Section 3: Primary antibody incubation Carefully...400 rpm for 2 h at room temperature or overnight at 4 °C . Section 5: TMB reaction Carefully remove the ...point. If your unknown sample’s absorbance falls above the range of the standard curve you will need to...

  7. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ...pH 8) 50 mM glucose 10 mM EDTA Store Solution I at 4°C Solution II - Denaturing Solution 0.2 N NaOH 1.0%...acetic acid 57 mL of dH 2 O Store Solution III at 4°C Grow 2 mL overnight cultures from single colonies...to the recovered aqueous DNA layer. Repeat steps 2-4. Note: Phenol-chloroform is a hazardous waste - DO...high speed (at least 12,000 rpm) for 15-30 min at 4°C. Notes: Pellet contains the precipitated DNA. Supernatant...temperature before quantifying and using. Store DNA at 4°C. Tips and FAQ Plasmid purification kits provide ... supernatant out of the tube if you are worried about losing the pellet. Dry with vacuum or by inverting...

  8. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ...Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or stable alternative, such...heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Polyclonal stable cell pool: see our protocol ...solution. Homogenized cell solution concentration: 4 × 10 5 cells/mL To seed one 96-well plate, make 10... of homogenized cell solution needed: (50 cells)/(4 × 10 5 cells/mL) = 0.125 µL Because this is such a...Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8):783-4. (Link opens in a new window) Last reviewed on: September...approximately 50–60% confluent. For 293T cells this is about 2 × 10 6 cells in a 10 cm dish. Each 10 cm dish ...cell solution into the conditioned medium prepared above to make a new cell solution at a concentration of...

  9. Lentivirus ddPCR Titration

    Type
    Protocol
    ...and transduce cells Day 4: Treat cells with Benzonase and harvest cells Day 4+: ddPCR and analysis Equipment...VIC) 1 µL 9 µL 900 nM, 250 nM Nuclease-free water 4 µL 36 µL Total Volume 16 µL 144 µL Vortex the master... collecting drops along the side of the tube. Add 4 µL of the 25 ng/µL samples to the appropriate PCR ...Extension 60 1 2 40 Enzyme Deactivation 98 10 2 1 Hold 4 ∞ 2 1 After PCR is complete, transfer the plate to...0.08366013072 6.69E+06 3 200 1620 15840 0.2045454545 8.18E+06 4 100 3180 20540 0.3096397274 6.19E+06 5 50 8960 17080...the titer. Calculations In the experimental setup above, the following virus dilutions were used to transduce...v = Virus volume, mL *In the experimental setup above, the cells per well is 300,000 and the virus volume...

  10. CRISPR Library Amplification

    Type
    Protocol
    ...2: Harvest cells and purify DNA (Estimated time 3-4 hours) Cells should be harvested first thing in the... Reagents 200 µL electrocompetent cells (Default: 4 tubes of Endura Duos, Lucigen, 60242-1) Alternatives...Agar + Antibiotic 65 mm Petri dish (VWR, 11019-552) 4 MaxiPreps (Qiagen HiSpeed Max, Catalog #12663) Tips...plates. Prechill Micropulser cuvettes on ice. Thaw 4 tubes of electrocompetent cells on ice for 15-20 minutes... incubation if needed. Place 100 mL sterile LB at 4 ℃. Day 2 (morning) Before beginning, prechill at least... LB-bacteria per plate. Centrifuge tubes (4000 G, 4 ℃, 15 minutes) to pellet bacteria. Decant LB and weigh...require modifications dictated by the originating laboratory for optimal results. If you obtained the pooled...

  11. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ...Prepare carbenicillin to a concentration of 1 mg/mL – 4 mg/mL in LB medium. The concentration of antibiotic... 1 mg/mL plate and effective selection. 150 µL of 4 mg/mL Carbenicillin plated over-agar Plate shows several...coli after Over-Agar Plating of Carbenicillin. The above graph displays the stock concentration of Carbenicillin...concentrations that will work for this assay, and the above result represents a single experiment. For publishable...

  12. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    ...Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or stable alternative, such...heat-inactivated FBS and 5mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and FBS lots can promote...Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8):783-4 (Link opens in a new window) . Last reviewed on: September...cells in the untransduced well (0 µL lentivirus, above) are dying. Perform regular media changes and monitor...

  13. Protocol - How to Inoculate a Bacterial Culture

    Type
    Protocol
    ...out the following into a 500 mL glass bottle: 4 g NaCl 4 g Tryptone 2 g Yeast Extract and dH 2 O to 400...suggested amount, instead of the other dry ingredients above. Media without growth (top) and with growth (bottom...

  14. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...gel with a pipette tip. Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for...will set more quickly if you place the gel tray at 4 °C earlier so that it is already cold when the gel...cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins. Optional...concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr... of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample... of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration ...

  15. AAV ddPCR Titration

    Type
    Protocol
    ...): 5 µL in 95 µL 1X PCR buffer (1:8,000) Dilution 4 (20X): 5 µL in 95 µL 1X PCR buffer (1:160,000) Dilution...Extension 60 1 2 50 Signal Stabilization 98 10 2 1 Hold 4 ∞ 2 1 After PCR is complete, transfer the plate to...factor of virus V = Volume of virus in reaction mix (4 µL) Sample Data When analyzing data there should be...dilution BSC. Prepare 1X dilution buffer (see recipe above). Save ~25 µL of 1X dilution buffer in a microcentrifuge...

  16. Protocol - How to Perform Sequence Analysis

    Type
    Protocol
    ...used to denote that the position could be any of the 4 bases. Check the trace file and see if you can manually...Addgene lists the primers used to obtain each result above the posted sequence in the "View Sequence" link....sequence than expected and wish to contact Addgene about the accuracy of your plasmid, please email help@...

  17. Water Bath Protocol

    Type
    Protocol
    ...walk-in refrigerators to achieve a temperature between 4°C and room temperature. This general protocol suits... lab! Introduction A water bath is a piece of laboratory equipment that helps bring your materials to ...even if you are using disinfectants as described above in step 3. You will also need to maintain the appropriate...

  19. Molecular Biology Protocol - Restriction Digest of Plasmid DNA

    Type
    Protocol
    ...cloning, it is recommended that you digest for at least 4 hours. If you will be using the digested DNA for another...more at (Link opens in a new window) NEB's website about star activity . If you are digesting a large number...

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

    Type
    Protocol
    ...therefore it is important that the digest go at least 4 hours and as long as overnight. If you are going to...Information about plasmid cloning by restriction enzyme digest (subcloning), including design and experimental...window) New England Biolabs for more information about restriction enzyme buffers). If you select enzymes...
Showing: 1 - 20 of 22 results