...oligos are cloned into the AgeI and EcoRI sites in place of the stuffer. The AgeI site is destroyed in most...one 7kb and one 1.9kb. Cut out the 7kb band and place in a sterile microcentrifuge tube. TIP: When visualizing... the media to remove the transfection reagent. Replace with 5 mL fresh DMEM + 10% FBS + penicillin/streptomycin...20 hours. Remove the virus-containing media and replace with fresh media. Do not add puromycin until at...should be worn at all times. Use plastic pipettes in place of glass pipettes or needles. Liquid waste should...

...at a lower voltage. You will want to have enough space around each band to cut without having DNA in other...Notes: To protect the UV box, it is a good idea to place the gel on a glass plate if available. Unlike the...than a certain total volume of gel per reaction. Place the gel in a labeled microfuge tube. Using a scale...

... from light. Procedure Section 1: Seeding cells Place a sterile poly-D-lysine coated coverslip in each...Alternative fixation methods such as methanol or acetone may be better for some applications. Remove the...mounting medium to the microscope slide. Gently place the coverslip on the microscope slide with the cell...

...dissolved in dH 2 O. *Carbenicillin can be used in place of ampicillin. Carbenicillin is more stable, so ...later if your forget your bottle in the autoclave. Place the gel mix in the autoclave and run on a setting...to allow them to dry. After overnight drying, we place the plates in a plastic bag with an absorbent material...

...Protocol Option #2) Water-saturated Phenol 2 M Sodium Acetate pH 4 Chloroform/Isoamyl alcohol (49:1) 75% Ethanol...hazards for these reagents. Work in a well-ventilated space and under a fume hood when working with the volatile...sequentially to 1 mL of lysate: Add 0.1 mL of 2 M sodium acetate (pH 4.0), mix thoroughly by inversion. Add 1 mL...

...until dried before overnight incubation if needed. Place 100 mL sterile LB at 4 ℃. Day 2 (morning) Before...transformation efficiency! Ensure that no arcing is taking place during electroporation. Arcing would manifest as...obtain sufficient intact pooled library even in the face of a recombined fraction but care must be taken ...

...cells AAV Purification by Iodixanol Gradient Ultracentrifugation Protocol Mulitchannel Pipetting Technique...personal protective equipment, setting up a clean workspace, and maintaining sterility while working. AAV ...

...Day 1: Transduce cells Day 2 (am): Remove media, replace with fresh media Day 4–5: Count fluorescent cells...Incubate for 48–72 h. Gently aspirate media and replace with 1 mL of PBS. Calculate the fraction of fluorescent-positive...

...2 (am): 18 h post transfection - Remove media, replace with fresh media Day 3 or more (am): Observe fluorescence...following morning, carefully aspirate the media. Replace the media with 15 mL of DMEM complete. Incubate...

... 2 (am): 18 h post-transfection. Remove media, replace with fresh media. Day 3-4 (am): Harvest virus Equipment...following morning, carefully aspirate the media. Replace the media with 10 mL of DMEM Complete or OptiPro...

...the lowest power objective to find your sample. Place your slide (or other sample type) on the microscope...stage. If using a slide, you can secure it into place using the metal clips on the stage. Turn on the ...

... is an excellent resource for choosing primers. Place thin-walled PCR tubes on ice Set up a 50 μL reaction...Reverse Primers DO NOT get added to a master mix. Place reaction tubes in PCR machine. Set annealing temperature...

...and transduce Cells Day 2–3 (am): Remove media, replace with fresh media containing selection reagent Day...important to monitor these cells regularly and replace the cell media. Cell death by some cells in the...

...versatile and allows for nearly any piece of DNA to be placed into a backbone vector of choice with minimal limitations... important to have nice crisp bands and to have space to cut out the bands. Because of this we recommend...

...remove the white sticker on the bottom of the gel. Place the gel in the electrophoresis chamber and secure...where dye is visible. Section 2: Staining the Gel Place the gel in a plastic tray with 100 mL of deionized...

...solvent. If using a NanoDrop to measure your samples, place 1-2µL of mini-prepped DNA onto the pedestal. Close...

...lid and the tube of a glycerol stock before you place the sample at -80°C. Frozen tubes are hard to write...

...that you can make a broad stroke. Only touch the surface of the plate, do NOT dig into the agar. Another...

...choice AAV Purification by Iodixanol Gradient Ultracentrifugation Purify adeno-associated virus from a preparation...

...electrophoresis . Tips and FAQ Restriction enzymes MUST be placed in an ice bucket immediately after removal from...