...details on doing diagnostic digests and how to interpret them please see the Diagnostic Digest page. Purifying... how to perform gel electrophoresis. Equipment Casting tray Well combs Voltage source Gel box UV light...the solution heats up.). Caution HOT! Be careful stirring, eruptive boiling can occur. Pro-Tip It is a good...

...mammalian antibiotic resistance (e.g., puromycin, blasticidin), which enables selection of a stable cell culture...untransduced cells, resulting in a more homogenous (but still polyclonal) cell population. Depending on the transducibility...Appropriate antibiotic for selection (e.g. puromycin, blasticidin) Reagent Preparation DMEM Complete: 10% v/v FBS...lentiCas9-Blast and then selected with 1 µg/mL blasticidin for 9 days. Single cells were then plated in...

...later. At a minimum we recommend the use of a diagnostic digest and high-throughput next generation sequencing...plasmid backbone a single time and visualize that digestion on an agarose gel ( see restriction digest protocol... Transform, recover, set up overnight growth (Estimated time 2-3 hours) Transformation should be performed...limited. Day 2: Harvest cells and purify DNA (Estimated time 3-4 hours) Cells should be harvested first...

...Insert Vector Type PI Publication Do you have suggestions for other plasmids that should be added to this...

...desired restriction sites and add them to your existing vector. It is a couple days of work that will ...the overhangs generated by restriction digest of existing sites in the original vector. Designing oligos...bases to complement the overhangs generated when digesting the vector with EcoRI and SalI (see diagram )....

...Equipment Water Bath 10% bleach or 70% ethanol Distilled water Disinfectant Thermometer Water bath weights...the edge of the slide. Fill the water bath with distilled water, not tap water, to prevent salts from accumulating...water in it from previous uses. However, you will still need to regularly clean out the water bath even ...

...percentage of fluorescent cells, these cells can still be used in downstream applications. Safety Warnings...ensure that you are in compliance with your institution’s biosafety regulations. Workflow Timeline Day...with multiple integration events leading to underestimation of the true titer. Calculate the transduction...

...genome-containing virions. Phenol red is added to clearly distinguish the steps. This method will help enrich the prep...ultracentrifugation. The steps are clearly distinguishable thanks to the use of phenol red. Image adapted...cartoon indicating the position of the needle for harvesting of the purified AAV using option #1. Right image...

... bath Pipetman Reagents 37 g pre-mixed powder consisting of: 5.0 g yeast extract 10.0 g peptone from casein...mL of agar and on which you can individually distinguish a maximum of ~100 colonies Pro-Tip If you will...After overnight drying, we place the plates in a plastic bag with an absorbent material to reduce condensation...

...lentiCas9-Blast 1 and then selected with 1 μg/mL blasticidin for 9 days. Single cells were then plated in ...transduced (MOI = 37) and selected with 1 μg/mL blasticidin for 9 days. Single cells from a stable cell pool...then expanded for an additional 3 weeks under blasticidin selection. Anti-Cas9 Western blotting was performed...

... May 2014 Vol. 15 No. 5, p. 3626 Do you have suggestions for other plasmids that should be added to this...

...ensure that you are in compliance with your institution’s biosafety regulations. Last Update: July 7, ... µg/mL. Mix well and incubate 72 h. Benzonase Digestion and Cell Harvest Gently aspirate media from the... When analyzing data there should be a clear distinction between negative droplets (black) and positive...

...for your eyes!), remove it from any gel tray as plastic will block much of the UV and with a clean, sterile... gel on a glass plate if available. Unlike the plastic tray, this will not significantly reduce the UV...

...Plasmid You may also like... How to Design a Primer Diagnostic Restriction Digest Introduction Sequence verification...the peaks in the area and make sure they are justifiable peaks. For instance, in the trace file below...

...recognition sequence. This can create multiple distinct overhangs with a single enzyme, and remove the...now look like this: Note: Elute your purified digestion and PCR products with sterile water (instead of...

...been placed in the centrifuge before you and may still be on the equipment. Before using the centrifuge...centrifuge. Immediately stop the centrifuge and investigate the issue. When you are finished spinning your...

...BSL-2). The purpose of the four levels is to distinguish between different types of hazards and to provide...with additional precautions to prevent injuries, ingestion, and exposures to hazardous materials. Video Watch...

...titers were similar to those reported by these institutions. This protocol is for a 96-well plate with 20...reaction volume. Last Update: February 13, 2019 Estimate of time required: ~3 h Workflow Timeline Plate...

...should be properly disposed of following your institution’s hazardous waste procedures. Last Update: February...desired concentration. If the sample concentration is still too low, repeat the centrifugation until the volume...

...ensure that you are in compliance with your institution’s biosafety regulations. Equipment Class II, Type... When analyzing data there should be a clear distinction between negative droplets (black) and positive...