.... Thaw a new vial of cells after 30 passages. Procedure Trypsinize and resuspend the HEK293T packaging...into 1 CF5. Return to incubator for 24 h - 36 h. Proceed with transfection: Calculate the amount of each...transfer supernatant to a sterile 500 mL bottle. Process the supernatant as follows: Filter through a 0.45...precipitation. Precipitation of the viruses can proceed overnight at 4 ℃ if needed. Transfer the entire...virus completely. Keep resuspended pellet on ice. Process the cell pellet as follows: Resuspend and lyse ...any residual DNA carried over from the packaging process. Incubate at 37 ℃ for 45 min. Transfer the viral...Transfer the clarified supernatant to a new tube and proceed with purification. Note: an aliquot of the solution...

...ordering process Ordering Page MTA FAQs Answers to frequently asked questions about the MTA process Help ...content, including how-to screen captures, lab procedure protocols, and career advice videos. Educational...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...Page MTA Guide - Requests Your guide to the MTA process when requesting samples from Addgene Technology...

...Libraries Molecular Biology Reference How do I process my Addgene pooled library? Introduction Please ...to autoclaved, sterile reagents for all steps. Procedure Day 1 Add 200 ng DNA to each 50 µL aliquot of ...not to split or gouge agar during the scraping process. Add each scrape into a 50 mL conical tube on ice...reagents (not including the recovery media!). Do not proceed with Maxipreps or NGS until adequate transformation...Visit our help center to get details on how to process your Addgene pooled library...

...DNA Ligation Introduction Transformation is the process by which foreign DNA is introduced into a cell.... 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...

... following your institution’s hazardous waste procedures. Last Update: February 7, 2022 Protocol Video... filter. Aliquot and freeze upright at -20 °C Procedure Section 1: Affinity chromatography Equilibrate...concentration of the pooled sample is above 1.0 mg/mL proceed to Option 1 with a buffer exchange using a Zeba...concentration of the pooled sample is below 1.0 mg/mL proceed to Option 2 with a buffer exchange/concentration...

...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, ...

...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 ...

...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...Centrifuge the culture to pellet the bacteria before proceeding with DNA preparation. *Pro-Tip* If your entire...

...video below to get a quick idea of how the whole process works. Equipment Autoclave Water bath Pipetman ...will grow). However, it is also more expensive. Procedure Measure 37g of pre-mixed LB-agar powder per L ...autoclave tape will darken during the autoclave process if your sample has spent at least 10 min at 121...

... suited for analyzing the gene’s function. The process is shown graphically in the following cartoon, ...TGCTTAGCGGCCGCTCAGTACTTCGAGATATGCCA-3’. Experimental Procedure Run PCR and purify the PCR product: Run PCR to...self-ligating recipient plasmid backbone. Transformation: Proceed with the transformation according to the manufacturer...

...completely to the template DNA strand so elongation can proceed. Usually a guanine or cytosine is used at the 3...creates primer dimers and disrupts the amplification process. When designing, if unsure about what nucleotide...

... (with appropriate antibiotic) Bacterial stab Procedure Obtain an LB agar plate with appropriate antibiotic...colonies. Once you have single colonies, you can proceed to Recovering Plasmid DNA or use the individual...

... and purify DNA fragments based on size. The procedure starts with standard agarose gel electrophoresis...concentration of the DNA that you isolated before proceeding to your next intended step with the now gel purified...

...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...

...together through the transformation/replication process. LIC employs long overhangs to form a stable association...free nucleotides from your PCR product before proceeding, as they will interfere with the exonuclease ...

...vector from ligating to itself during the ligation process. If the sticky ends on either side of the vector...ligation can be improved by incubation at 37°C. Proceed with bacterial transformation . Tips and FAQ Do...

...polyethyenimine (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...

...aliquot and store at -80 ℃ for up to one year. 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% ...

...filter. Aliquot and freeze upright at -20 °C. Procedure Section 1: Seeding cells The day prior to transfecting...between 1.5–2 x 10 6 cells/mL with >95% viability to proceed with transfection. Transfect the flask containing...

... be subjected to multiple freeze-thaw cycles. Procedure Before beginning, determine the optimal dose of...antibiotic. This is the beginning of the selection process, which will begin the selection of a stable cell...