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We narrowed to 34 results for: mme

Showing: 1 - 20 of 34 results

  1. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...efficiency of the virus, so Addgene recommends that you use the virus immediately or aliquot the media into smaller...pLKO.1 C. Cloning shRNA oligos into pLKO.1 C.1 Recommended materials C.2 Annealing oligos C.3 Digesting ...transforming into bacteria D. Screening for Inserts D.1 Recommended materials D.2 Screening for inserts E. Producing...Producing Lentiviral Particles E.1 Recommended materials E.2 Protocol for producing lentiviral particles F... F. Infecting Target Cells F.1 Recommended materials F.2 Determining the optimal puromycin concentration...following plasmids available from Addgene are recommended for use in conjunction with the pLKO.1 TRC-cloning...mismatches to all unrelated genes. TIP: Addgene recommends that you select multiple target sequences for...

  2. CRISPR Library Amplification

    Type
    Protocol
    ...(1x10 10 cfu/µg) commercially prepared electrocompetent cells is strongly recommended. 800 ng pooled library... pellets for later purification. Immediately purify them! Commercial Maxipreps rely on incremental, ordered...specific pooled library available at Addgene. We recommend following the depositor's specific protocol if... headaches and expense later. At a minimum we recommend the use of a diagnostic digest and high-throughput...specifications described in the equipment manual. Immediately add 1 mL SOC to cuvette. Remove all liquid from...

  3. Pouring LB Agar Plates

    Type
    Protocol
    ...Antibiotic Concentrations Antibiotic Recommended Stock Concentration Recommended Working Concentration Ampicillin...will be screening a large number of colonies, we recommend using larger plates. Many labs use 100 mm x 15...over at high temperature. Pro-Tip Although our recommended temperature should kill most potential contaminants...Pro-Tip Once your autoclave cycle is complete, we recommend opening the door to the autoclave just a crack...certain your agar is at the right temperature, we recommend using a laser thermometer. Light the flame at...

  4. Molecular Biology Protocol - Restriction Digest of Plasmid DNA

    Type
    Protocol
    ...aliquot of your plasmid of interest (see below for recommend amounts) Appropriate restriction enzyme (see manufacturer's...following: DNA Restriction Enzyme(s) Buffer BSA (if recommended by manufacturer) dH 2 O up to total volume Pro-Tips...Restriction Enzyme 3 µL 10x Buffer 3 µL 10x BSA (if recommended) x µL dH 2 O (to bring total volume to 30µL) ...digests with >1 µg of DNA used for cloning, it is recommended that you digest for at least 4 hours. If you ...Restriction enzymes MUST be placed in an ice bucket immediately after removal from the -20 °C freezer because...

  5. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...Master Mix 2X Primer pair targeting AAV2 ITR ( Aurnhammer et al., 2012 ) fwd ITR primer, 5'-GGAACCCCTAGTGATGGAGTT...forward and reverse primers. Vortex briefly, immediately before use. Use a reservoir and a multichannel...gave reliable and consistent results. Use the recommended plasmid, or test multiple plasmids to find a ...internal reference virus that is 1 x 10 13 GC/mL. We recommend always using a reference within 1-log of the expected... Each curve represents a dilution. References Aurnhammer C, Haase M, Muether N, Hausl M, Rauschhuber C...

  6. Protocol - How to Perform Sequence Analysis

    Type
    Protocol
    ... confirm. Addgene's plasmid information pages recommend 5’ and 3’ sequencing primers. These primers typically...sequence the entire plasmid. Addgene strongly recommends that you sequence any portion of the plasmid ... 300 nucleotides from your target. Are there commercially available primers that can be used for sequencing...sequencing and need to design a custom primer, Addgene recommends using Addgene's sequencing results as a reference...

  7. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ...new window) NEB for more information). Thus, we recommend that you add 3-6 bases upstream of your restriction...important to digest plenty of starting material. We recommend using your entire PCR reaction and 1μg of recipient...space to cut out the bands. Because of this we recommend that you use a wide gel comb, run the gel on the...fuse your insert to your recipient plasmid. We recommend around 100ng of total DNA in a standard ligation...

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

    Type
    Protocol
    ...purify your DNA away from the agarose using a commercially available kit or standard protocol. Anneal oligos...EDTA) and mixed in equimolar concentrations. We recommend mixing 2μg each in a total volume of 50μL - add...oligos in a PCR tube. Place tube in a thermocycler programmed to start at 95°C for 2 minutes. Then, gradually...

  9. Protocol - Bacterial Transformation

    Type
    Protocol
    ...For the highest transformation efficiency, we recommend that you follow the instructions that came with...with your competent cells. Pro-Tips Commercial competent cells range significantly in their transformation...containing the appropriate antibiotic. Pro-Tips We recommend that you plate 50 μL on one plate and the rest...

  10. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...to use of your purified virus. Nonetheless, we recommend performing a buffer exchange before using the ...QuickSeal tube at the bottom using an 18 ga needle. Immediately start collecting 0.5 to 1 mL fractions in microcentrifuge...that has settled at the bottom of the column. We recommend concentrating to a minimum of 500 µL. If the concentrate...

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

    Type
    Protocol
    ...important to digest plenty of starting material. We recommend 1.5-2μg of donor plasmid and 1μg of recipient ...space to cut out the bands. Because of this we recommend that you use a wide gel comb, run the gel on the...fuse your insert to your recipient plasmid. We recommend around 100ng of total DNA in a standard ligation...

  12. AAV ddPCR Titration

    Type
    Protocol
    ... reduce the risk of contaminating reagents we recommend making small aliquots of master mixes, primers...Buffer containing 0.05% Pluronic F-68: Prepare immediately before use and vortex before using. 500 µL of... > 10 to be invalid. To reduce NTC values, we recommend wiping down all pipettes and equipment with 10%...

  13. Lentivirus ddPCR Titration

    Type
    Protocol
    ...of each sample. Samples can be used for ddPCR immediately or stored at -20 °C until ready to use. Preparing...infectious titer. Tips and Troubleshooting We recommend wiping down all pipettes and equipment with 10%... reduce the risk of contaminating reagents we recommend making small aliquots of master mixes, and primers...

  14. Protocol - How to Perform a Diagnostic Digest

    Type
    Protocol
    ...given sequence. For a list of the commonly used commercially available restriction enzymes, see New England...aliquot of your plasmid of interest (see below for recommended amounts) Appropriate restriction enzyme (see ...

  15. Fluorescence Titering Assay

    Type
    Protocol
    ...Titer will vary between cell lines. It is not recommended to subject lentiviral supernatants to multiple...you are freezing and aliquoting virus, it is recommended that you titer from the frozen stock to account...

  16. Lab Safety for Biosafety Levels One and Two

    Type
    Protocol
    ... be done underneath a hood. Clean any spills immediately, and decontaminate as necessary. For large spills...bloodborne pathogens training. It is strongly recommended that anyone participating in BSL-2 work receives...

  17. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...digestion . Most restriction enzymes digest DNA asymmetrically across their recognition sequence, which results... 1 vector molar ratio will work just fine. We recommend around 100ng of total DNA in a standard ligation...

  18. Immunocytochemistry

    Type
    Protocol
    ...between products. Addgene does not endorse or recommend specific products or equipment. Inclusion of this...antibody concentrations, incubation times, and recommended compatible reagents. Secondary antibodies must...

  19. Colony Formation Titering Assay

    Type
    Protocol
    ...Titer will vary between cell lines. It is not recommended that lentiviral supernatants be subjected to ...you are freezing and aliquoting virus, it is recommended that you titer from the frozen stock to account...

  20. Coomassie Purity Stain of Recombinant Antibodies

    Type
    Protocol
    ...between products. Addgene does not endorse or recommend specific products or equipment. Inclusion of this...water IgG isotype standard 2.5 mg/mL - can use commercial standard or validate an in-house standard Before...
Showing: 1 - 20 of 34 results