... for all steps. Procedure Day 1 Add 200 ng DNA to each 50 µL aliquot of thawed Endura Duos on ice. Flick...representation of individual plasmids in the pooled library during amplification. This protocol is designed to be ... µL electrocompetent cells (Default: 4 tubes of Endura Duos, Lucigen, 60242-1) Alternatives include Stbl4... cm cuvette, 1.8 kV, 1 pulse. Aliquot 25 µL DNA-Endura into pre-chilled cuvette. Pulse. Caution Electroporation...contain a total of 5 mL (3 mL SOC + 2 mL transformed Endura from two separate transformations). Pro-Tip Do ...mix when removing SOC containing transformed DNA-Endura from cuvette. Shake four 14 mL Vented Falcon Tubes...pulling motion Take care not to split or gouge agar during the scraping process. Add each scrape into a 50...

...polyethylenimine (PEI) transfection protocol. This procedure can be modified for alternative packaging cell...that are below passage 15 for viral production. Procedure Seed 293T packaging cells at 3.8×10 6 cells per...DNA tube. Incubate the mixture 12–15 min at RT. During the incubation, add 10 mL of DMEM Complete to a... pellet any packaging cells that were collected during harvesting. Filter supernatant through a 0.45 μm...

...glucose, lower FBS media causes less filter clogging during harvesting. The addition of sorbitol has been shown.... Autoclave or sterile filter. Pro-Tip Stirring during the cooling period is recommended or the solution...overgrow your cells. Pass the cells twice a week during the maintenance phase and do not allow cells to.... Thaw a new vial of cells after 30 passages. Procedure Trypsinize and resuspend the HEK293T cells from...

...isolate and purify DNA fragments based on size. The procedure starts with standard agarose gel electrophoresis...with the razor blade. This is especially important during the DNA purification step, as many kits cannot ...used to determine how much of each buffer to add during the DNA isolation step. Finally, you will want ...

...content, including how-to screen captures, lab procedure protocols, and career advice videos....Video protocol guides for standard laboratory procedures. How-To Videos Screencapture walk-throughs to...Gel electrophoresis is the standard laboratory procedure for separating DNA by size for visualization and...

...densities is essential to many different experimental procedures, such as minipreps and RNA extractions. Plus,...temperatures. This protocol will cover the general procedure and features to keep in mind when using any centrifuge...the time and speed needed for your experiment. Procedure An example of a balanced tabletop centrifuge, ...

... competent cells. This is a relatively simple procedure and is useful for performing low efficiency transformations...media Competent cells DNA you'd like to transform Procedure Take competent cells out of -80°C and thaw on ...competent cells) to ensure that your transformation procedure is working. TIP: Sometimes less is more. Although...

...of viral transduction, and not during the cell seeding step. Procedure Before beginning a colony formation...cells and debris. Lentiviral titer can decrease during cycles of freeze-thaw. If you are freezing and ...

...Introduction Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base...Ethidum bromide (stock concentration of 10 mg/mL) Procedure Pouring a Standard 1% Agarose Gel: Measure 1 g... from Your Gel: If you are conducting certain procedures, such as molecular cloning, you will need to ...

...subcloning), including design and experimental procedures....will save you time in future steps. Experimental Procedure Digest your DNA Set up restriction digests for...and recipient plasmids. Because you lose some DNA during the gel purification step, it is important to digest...

...resistance gene (or other antibiotic resistance) Procedure Day 1 Prepare carbenicillin to a concentration... give the antibiotic time to more fully absorb. During the incubation, transform DH5α E. coli cells by...

...to add short tags or shRNAs to any vector (the procedure will simply differ in terms of primer design)....be performed enzymatically later. Experimental Procedure Digest and purify vector While waiting for your...

....) Reagents Material that will be weighed out Procedure The balance in the photo above has a capacity ...your container. Make sure that any spills produced during the protocol are cleaned up. Discard the weighing...

... supernatants to multiple freeze-thaw cycles. Procedure Seed 75,000 cells into each well of a 6-well dish...cells and debris. Lentiviral titer can decrease during cycles of freeze-thaw. If you are freezing and ...

...filter. Aliquot and freeze upright at -20 °C. Procedure Section 1: Seeding cells The day prior to transfecting...Section 3: BCD Feed and valproic acid supplementation During the 24–144 h post-transfection, supplement the ...

...of Buffer B and 2 mL of 5 M NaCl to 43 mL PBS Procedure Preparation and loading of the iodixanol gradient...0.001% in PBS: 45 mL PBS + 5 ml B + 200 mM NaCl Procedure Cover the filter membrane with 15 mL of 0.1% Pluronic...

...plasmid DNA. This page will discuss the general procedure for purifying plasmid DNA from bacterial culture...protocol below is meant to describe the general procedure for purifying plasmid DNA from bacterial cultures...

...Polymerase (5 units/μL) PCR Machine Agarose Gel Procedure Primer Design and PCR Design Primers. See our ... that can withstand radical temperature changes during a typical PCR. The DNA polymerase has an optimum...

...0.15 μL 15 μL Nuclease Free Water 4.7 μL 470 μL Procedure Prepare a plasmid stock of 2 x 10 9 molecules/...amount of background signal that is most evident during initial PCR cycles. This background signal must...

...will grow). However, it is also more expensive. Procedure Measure 37g of pre-mixed LB-agar powder per L ... autoclave tape. The autoclave tape will darken during the autoclave process if your sample has spent ...