...21028 Protein G GraviTrap columns, Cytiva 28-9852-55 or rProtein A GraviTrap columns, Cytiva 28-9852-54 LabMate...Desalting Columns, 7 kDa MWCO, 10 mL, Thermo Fisher 89893 Amicon Ultra-15 30 kDa MWCO column, Millipore...Reservoirs to the top of the Gravitrap column. Pro-Tip Gravitrap columns have a finite binding capacity. If...among multiple columns. For information about the total binding capacity for your columns, refer to the...Gravitrap column to a clamp on a clamp stand. Use scissors to cut the bottom of the Gravitrap column at the...Gravitrap columns at the bottom. Add 5 mL of Pierce IgG Elution Buffer to the capped Gravitrap column and incubate...Amicon 30 kDa MWCO buffer exchange column. (Optional) Regenerate the column by washing with 25 mL of Protein...

...liquid into the pipette tip, the volume display which shows the set volume for the the pipette, and the tip...shows the volume adjustment ring right below the plunger. This ring changes the pipette volume. Below that...with and the volume to be dispensed. The boxes that the tips come in often indicate a volume range that ...shows the pipette with the appropriate dispense volume: This image shows the P2, P10, P20, P100, P200,...P1000 as shown from left to right. Pipette Dispense Volume P2 0.2 to 2 µL P10 1 to 10 µL P20 2 to 20 µL P100...be closed to prevent contamination. Reading the Volume on the Pipette Although each pipette performs the...picture above), while 0.5 µL would read as 050. Each volume display on these pipettes will also have small ...

... below. Dilution Factor Virus Volume to Dilute Media Volume Total Volume 2.5 160 µL viral stock 240 µL...enough master mix for 9 samples. Component Volume 9X Volume Final Concentrations 2X ddPCR Supermix for...per well is 300,000 and the virus volume refers to the total volume the cells were transduced, which is...microcentrifuge tubes with the appropriate amount of media volume listed in the chart below. For the first virus ... well with a 1 mL pipette 5–10 times. The final volume in the well is 1.5 mL, and the final Polybrene ...nM, 250 nM Nuclease-free water 4 µL 36 µL Total Volume 16 µL 144 µL Vortex the master mix for 15 sec and... # Cells per well D = Dilution factor v = Virus volume, mL *In the experimental setup above, the cells...

...cell suspension (no clumps of cells). Pool cells from 2 x T-175 flasks. Adjust volume to 300 mL with DMEM...filter system, Nalgene, 565-0010 (or for larger volumes 1000 mL 0.45 µm Rapid-Flow PES Filtration Unit,...NaCl into deionized water and adjust to a final volume of 1000 mL. Stir at RT until fully dissolved. Pro-Tip...vigorously to obtain a single cell suspension (no clumps). Count cells using a hemocytometer or cell counter...million cells from the CS2 into one CS5 with a total volume of 850 mL. Return to the incubator for 24 h–36 ...Plasmid Plasmid Size (bp) DNA Concentration (μg/μL) Volume of DNA (μL) RepCap 7,265 1.00 727.6 pHelper 11,...Calculations RepCap: 0.08 μg/bp × 7,265 bp = 582.1 μg Volume Needed: 582.1 μg / 1.0 μg/μL = 582.1 μL pHelper...

...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...-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...

...cell pellet in appropriate volume of cold lysis buffer. Pro-Tips The volume of lysis buffer will vary ...Nonfat milk powder 96-well microtiter plate Chemiluminescence substrate Plastic wrap Primary antibody Secondary...the sample from the standard curve. Determine the volume of sample required to load an equivalent amount... at RT on a shaking platform. Prepare the chemiluminescence substrate by mixing 1:1 reagent A to reagent...reagent B. Gently incubate the membrane in the chemiluminescence reagent for 5 min at RT . Cover the membrane...with clear plastic and use a gel imager with chemiluminescence detection or a dark room to detect the bands...

...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 Method 2 $$T = {N...fluorescence D = Dilution Factor V T = Transduction Volume, mL For example: If 150,000 cells were transduced...Fraction of cells with fluorescence V V = Virus Volume, mL For example: If 15 µL of virus added to 150,000...

...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...

...stock made in step #1): Volume of 2 x 10 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 Update: February 13, 2019 Estimate of time...least 10 times, and use at least half of the final volume (mix with >50 uL if your well contains 100 uL) ...

... 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 dilution of the homogenized...transferring 0.125 µL, now transfer 100 times that volume, which is 12.5 µL. To make the final 5 cell/mL ...

...recommended by manufacturer) dH 2 O up to total volume Pro-Tips 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. A typical restriction digestion reaction...BSA (if recommended) x µL dH 2 O (to bring total volume to 30µL) The amount of restriction enzyme you use... enzyme because it is difficult to pipette less volume than this; 0.2-0.5 µL will likely be more enzyme...

... 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...

.../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...

...enough master mix for 9 samples. ITR Master Mix Volume 9X Volume Final Concentration 2X ddPCR Supermix for .../mL R = Reaction volume (20 µL) C = Copies/µL D = Dilution factor of virus V = Volume of virus in reaction...900 nM Nuclease-free water 5.9 µL 71.55 µL Total Volume 20 µL Vortex the master mix for 15 sec and spin...

...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...

.... 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 ...

...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. ...

... 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 ...

...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 ...

...1 1 μL AgeI add ddH 2 O to bring to 50 μL final volume Incubate at 37°C for 2 hours. Purify with Qiaquick... DNA ligase add ddH 2 O to bring to 20 μL final volume Incubate at 16°C for 4-20 hours. Transform 2 μL...0.8 μL NcoI add ddH 2 O to bring to 20 μL final volume Incubate at 37°C for 1-2 hours. 4. Run the digestion...master mix to each tube from step c for a total volume of 100 μL. Pipette master mix directly into the...