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Showing: 1 - 20 of 28 results
  1. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ...solution to a column that will bind the now precipitated DNA. Wash the pellet or column with 70% ethanol...your DNA solution, add 2-2.5 volumes 95% or 100% ethanol and 1/10 volume of 3 M Na-acetate (pH 4.8). Invert...Pro-Tip* Refer to appropriate DNA prep protocol for volume of bacteria to grow (low copy plasmids require ...bacteria back into suspension, but within a smaller volume of buffer that is compatible with the next solution...Resuspend the DNA pellet, or elute the DNA off of the column using water or a neutral buffer such as TE. You...Phenol-Chloroform Extraction of DNA Samples Add an equal volume of TE-saturated phenol-chloroform to the aqueous...and place it in a new microfuge tube. Add equal volume of chloroform to the recovered aqueous DNA layer...
  2. AAV Production in HEK293 Cells

    Type
    Protocol
    ...cell suspension (no clumps of cells). Pool cells from 2 x T-150 flasks. Adjust volume to 200 mL with DMEM...single cell suspension (no clumps). Add 435 mL of complete medium (final volume 500 mL). Seed 250 millions...24 g of NaCl into ddWater and adjust to a final volume of 1,000 mL. Stir at room temperature until fully...Plasmid Plasmid Size (bp) DNA Concentration (μg/μl) Volume of DNA (μl) RepCap 7,265 1.00 727.6 pHelper 11,...Calculations RepCap: 0.10 μg/bp × 7,265 bp = 727.6 μg Volume Needed: 727.6 μg/ 1.0 μg/μl = 727.6 μl pHelper:...pHelper: 0.10 μg/bp × 11,854 bp = 1185.4 μg Volume Needed: 1185.4 μg/ 1.0 μg/μl = 1185.4 μl Transfer Plasmid...Plasmid: 0.10 μg/bp × 5,842 bp = 584.2 μg Volume Needed: 584.2 μg/ 1.0 μg/μl = 584.2 μl Aliquot 176 mL of OptiMEM...
  3. Lentivirus ddPCR Titration

    Type
    Protocol
    ...dilutions. Dilution Factor Virus Volume to Dilute Media Volume Total Volume 2.5 160µL viral stock 240µL 400µL...enough master mix for 9 samples. Component Volume 9X Volume Final Concentrations 2X ddPCR Supermix for...each well with a 1mL pipette 5-10 times. The final volume in the well is 1.5mL and the final Polybrene concentration... 900nM/250nM Nuclease-free water 4µL 36µL Total Volume 16µL 144µL Vortex the master mix for 15s and spin.../genome)*(#cells/well)*(dilution factor)/(virus volume) Take the average infectious titer obtained from...
  4. Fluorescence Titering Assay

    Type
    Protocol
    ...dilutions. Dilution Volume of Lentivirus Stock (μL) Volume of DMEM complete (μL) Volume of 10mg/mL polybrene...using either the dilution factors (method 1) or the volume of virus (method 2): Method 1 : TU/mL = (Number...Percent fluorescent x Dilution Factor)/(Transduction Volume in mL) Method 1 example : If the 1:100 well has... cells transduced x Percent fluorescent)/(Virus volume in mL) Method 2 example : If 15 µL of virus added...
  5. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...bottom of the column. We recommend concentrating to a minimum of 500 µl. If the concentrate volume is less ...video below to learn setup and use an iodixanol column gradient for AAV purification. Workflow Timeline...MgCl 2 and 14.91 mg of KCl in 1× PBS in a final volume of 100 mL. Sterilize by passing through a 0.22-...MgCl 2 , and 14.91 mg of KCl in 1× PBS in a final volume of 100 mL. Sterilize by passing through a 0.22-...less than 500 µl, bring up the volume with formulation buffer. Use a P1000 to the bottom of the filter and...
  6. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...stock made in step #1): Volume of 2x10 9 stock or previous dilution (μL) Volume of nuclease-free water ... in the table below: Dilution Series Volume of sample(uL) Volume of nuclease free water (uL) Dilution ...protocol is for a 96-well plate with 20 μL reaction volume. Last Modified: February 13, 2019 Estimate time...least 10 times, and use at least half of the final volume (mix with >50uL if your well contains 100uL) Use...
  7. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ... Determine the volume of homogenized cell solution that corresponds to 50 cells: Volume of homogenized...cell pool by trypsinization and break up any cell clumps into individual cells by passing several times ...conditioned medium, it will be more accurate if a higher volume of homogenized cell solution is transferred. Transfer...cells/mL) = 0.125 µL Because this is such a small volume, first make 1 mL of a 1:100 diliution of the homogenized...transfering 0.125 µL, now transfer 100 times that volume, which is 12.5 µL. To make the final 5 cell/mL ...
  8. Molecular Biology Protocol - Restriction Digest of Plasmid DNA

    Type
    Protocol
    ...recommended by manufacturer) dH 2 O up to total volume *Pro-Tip* The amount of DNA that you cut depends... often requires 1 µg of DNA. The total reaction volume usually varies from 10-50 µL depending on application...application and is largely determined by the volume of DNA to be cut. *Pro-Tip* A typical restriction digestion...BSA (if recommended) x µL dH 2 O (to bring total volume to 30µL) *Pro-Tip* The amount of restriction enzyme... enzyme because it is difficult to pipette less volume than this; 0.2-0.5 µL will likely be more enzyme...
  9. General Transfection

    Type
    Protocol
    ... 2 flask in a volume of 15 mL. Wednesday: Plate 1x10 6 cells in a T75 flask in a volume of 15 mL. Friday...Friday: Plate 8x10 5 cells in a T75 flask in a volume of 15 mL. Do not add antibiotics to the media. Use...depending on the user, quantities of chemical used, volumes, pH adjustment etc. Consequently, each batch needs...ratios to test: Ratio of DNA:PEI Amount of DNA (μg) Volume of 1 mg/mL PEI (μL) 1:1 18.9 18.9 1:2 18.9 37.8...
  10. Colony Formation Titering Assay

    Type
    Protocol
    .../mL polybrene: Dilution Volume of Lentivirus or Previous Dilution (μL) Volume of DMEM Complete Containing...Containing 10 μg/mL Polybrene (μL) Volume of Virus Added to Plate (μL) Volume of Cells Added to Plate (μL) ...mL) as follows: TU/mL = number of colonies/total volume in the well (mL) x dilution factor e.g., If the...
  11. AAV ddPCR Titration

    Type
    Protocol
    ...enough master mix for 9 samples. ITR Master Mix Volume 9X Volume Final Concentration 2X ddPCR Supermix for ...1000/V)]*D} T = GC/mL R = reaction volume (20µL) C = Copies/µL V = volume of virus in reaction mix (4µL) ...16.2µL 900nM Nuclease-free water 5.9µL 71.55µL Total Volume 20µL Vortex the master mix for 15 seconds and spin...
  12. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    .... Mix the dilutions well. Dilution Volume of Lentivirus (μL) Volume of DMEM complete + 10 µg/mL polybrene...cells as follows: Dilute 350,000 cells into a total volume of 7 mL of DMEM complete + 10 µg/mL polybrene. ... well of the 6-well dish. This brings the total volume in each well up to 1.5 mL. Since all the media ...
  13. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...master mix,” which minimizes the number of small volumes you need to pipet. If you are using the same template...PCR product. Master Mix Preparation Multiply the volume of each reagent by the number of individual PCRs...reactions (7 unique primer pairs), so we multiply each volume by 7. In a single 1.5mL tube combine the following... 2.5µl (fwd primer) – 2.5µl (rev primer) = 45µl volume of master mix to add to each PCR tube. Secure the...
  14. Kit Free RNA Extraction

    Type
    Protocol
    ...disturbing the interphase layer, consider using a lower volume pipettor like a p200 to collect a majority of the...Chloride for this step. Isopropanol (Option A) - Add 1 volume of Isopropanol to the extracted aqueous layer. ...
  15. Pooled Library Amplification

    Type
    Protocol
    ... the Maxiprep protocol, scale the reagent volume and column number as needed. The use of Mega or Gigapreps... plateau and sometimes fall off entirely if the column or reagents are significantly overloaded. If you...Consult the manufacturers handbooks for appropriate volumes and numbers of tandem purifications. Visit our ...
  16. Protocol - How to Purify DNA from an Agarose Gel

    Type
    Protocol
    ...many kits cannot handle more than a certain total volume of gel per reaction. Place the gel in a labeled... the gel is directly proportional to its liquid volume and this is used to determine how much of each ...sample you want to load on a gel, make 10% more volume than needed because several microliters can be ...
  17. Pouring LB Agar Plates

    Type
    Protocol
    ...200 mL of agar in 500 mL bottles. The extra empty volume is necessary to prevent your molten agar from boiling...Cover the opening of the bottle with its cap or aluminum foil (but do not make an air-tight seal!) and ...the first plate to get a good idea of what that volume looks like in your particular plate. For the remainder...
  18. Protocol - How to Inoculate a Bacterial Culture

    Type
    Protocol
    ...loosely cover the entire top of the bottle with aluminum foil. Autoclave and allow to cool to room temperature... swirl. Loosely cover the culture with sterile aluminum foil or a cap that is not air tight. Incubate ...
  19. Ligation Independent Cloning

    Type
    Protocol
    ...Inactivate T4 Pol by heating to 75° for 20 minutes. Volume (μl) Reagent Final Conc. 4 10X Buffer 1X 20-30 ...annealing reaction. This should be done in a small volume with no additional water (<5 μl). It is advisable...
  20. Protocol - Bacterial Transformation

    Type
    Protocol
    ...recover all transformants. *Pro-Tip* If the culture volume is too big, gently collect the cells by centrifugation...centrifugation and resuspend in a smaller volume of LB so that there isn't too much liquid media on the agar...
Showing: 1 - 20 of 28 results