...Iodixanol Gradient Ultracentrifugation AAV Purification by Iodixanol Gradient Ultracentrifugation You may also...tube at the interface of the 60% and 40% gradient (see Figure 2) with an 18 ga needle. Place the first ...fractions per tube. Avoid the proteinaceous material near the 40–25% interface. *Pro-Tip* As soon as the ...care to avoid collecting the proteinaceous material at the 40–25% interface. Repeat for each QuickSeal ...Iodixanol gradient after ultracentrifugation. The arrow indicates the 60–40% interface. The vertical black ...Follow this protocol using Iodixanol gradient ultracentrifugation to get pure adeno-associated virus (AAV) ... be completed in one (long) day. Equipment Ultracentrifuge T70i rotor or equivalent Reagents Opti-Prep...

...the trace file, you will see that these base calls are unreliable. This is an example of a trace file...with the sequence, trace file, and primer used. What program can I use to view my trace file? There are ...You should receive your sequencing results as a trace file (.ab1) which graphically depicts the sequence...four nucleotide bases. This is an example of a trace file from a high-quality portion of a sequencing...and include: The sequencing result The original trace file (.ab1 file) provided by your sequencing company...sequencing result, what should I do? Check your trace file first; the apparent mismatch/mutation may be... be the result of a mis-called peak in the trace file. If the mutation is not an artifact, please email...

...bath when they are placed inside. Water bath floats can be used to secure tubes in place and water bath weights... hold bottles in place. After putting your tubes or bottles in the water bath, place the lid back on. ...bottles and containers. Water baths can also be placed in walk-in refrigerators to achieve a temperature...water, to prevent salts from accumulating on the surfaces of the water bath as the water evaporates from...bottle, for example, number of drops per liter. Place a thermometer in the water bath. Oftentimes, the...bath will have a spot to secure the thermometer in place. Turn the water bath on and set the appropriate ... Water baths tend to heat up relatively slowly. Place the water bath cover on the top of the water bath...

...mini centrifuge and place on ice. Wipe down a DG8 cartridge holder with bleach and place in the Biological...Serial Dilution Place a 48-well dilution plate in a chilled 96-well freezer block and place in the dilution...temperature is reached, place the PCR plate with the foil onto the metal support block. Place the block in the... on ice before use. Wipe down all pipettes and surfaces with 10% bleach. Safety Warnings AAV is generally... BSC until ready to use. Prepare the Master Mix Place a ddPCR plate onto a chilled 96-well freezer block...spin in a mini centrifuge for 10 sec before use. Place an 8-well PCR tube strip into a chilled 96-well ...1X dilution buffer from step 9 to the NTC tube. Place a DG8 cartridge into the cartridge holder. Using...

...running buffer and 5 μL of EtBr, place on a rocker for 20-30 mins, replace EtBr solution with water and destain...the agarose into a gel tray with the well comb in place. *Pro-Tip* Pour slowly to avoid bubbles which will... the sides/edges of the gel with a pipette tip. Place newly poured gel at 4 °C for 10-15 mins OR let sit...in a hurry, the gel will set more quickly if you place the gel tray at 4 °C earlier so that it is already...instead of diffusing in the buffer. Once solidified, place the agarose gel into the gel box (electrophoresis... you had not added EtBr to the gel in the first place. Carefully load a molecular weight ladder into the...prevent bubbles or buffer from entering the tip. Place the very top of the tip of the pipette into the ...

...pellets form in approximately the same place in each sample. Place the centrifuge’s internal lid on the ... appropriate for the biosafety level of the lab space Reagents Sample to be centrifuged Water Background...cousins, but can hold much larger containers. Ultracentrifuges are also generally floor sized centrifuges...the lid open, the rotor with microfuge tubes in place, and the control panel where you can set the time...picture above. Wear PPE appropriate for the lab space in which you are working. Even if your samples may...specific PPE, you never know what else has been placed in the centrifuge before you and may still be on...that everything appears to be working smoothly. Place your sample tubes into the rotor so that they are...

... Stack in the plastic tray. Place the Bottom Stack on the blotting surface. Align the electrical contacts...blotting surface of the iBlot 2 Gel Transfer Device. Wet the pre-run gel in deionized water and place it on... Load 5–10 µL of the prestained protein ladder. Place the lid on the tank and plug the electrode cords...piece of iBlot Filter Paper in deionized water. Place the pre-soaked iBlot Filter Paper on the gel and...and remove the air bubbles with the roller. Place the Top Stack over the pre-soaked filter paper. Remove...Remove air bubbles using the roller. Place the Absorbent Pad on top of the Top Stack such that the electrical...corresponding electrical contacts on the blotting surface of the iBlot 2 Gel Transfer Device. Close the lid...

...mini centrifuge and place on ice. Wipe down a DG8 cartridge holder with bleach and place it in the Biological...temperature is reached, place the PCR plate with the foil onto the metal support block. Place the block in the... on ice before use. Wipe down all pipettes and surfaces with 10% bleach. Safety Warnings Lentivirus is...touching the screen. Preparation of the Master Mix Place a ddPCR plate onto a chilled 96-well freezer block...spin in a mini centrifuge for 10 sec before use. Place an 8-well PCR tube strip into a chilled 96-well ... forth 10 times to mix. Generating the Droplets Place a DG8 cartridge into the cartridge holder. Using...dispensing the droplets down the side of the tube. Place a Pierceable Foil Heat Seal on the PCR plate with...

...Renaturing Solution (Potassium Acetate) 120 mL 5M Potassium acetate 23 mL glacial acetic acid 57 mL of dH 2 O Store...volume of 3 M Na-acetate (pH 4.8). Invert the microfuge tube to mix. (Optional) Place the tube either ...isopropanol 90% ethanol 70% ethanol TE buffer 3 M Na-acetate (pH 4.8) Protocol: Generalized DNA Purification...phase (protein) Pipet the aqueous DNA layer and place it in a new microfuge tube. Add equal volume of ...

...accuracy as these types of scales display more decimal places. *Pro-Tip* If you’re weighing out an amount larger... the sucrose in your container after each time. Place the weighing boat or weighing paper onto the balance...fold it in half first to create a crease and then place it unfolded onto the balance. This will help you... on as otherwise, the paper could stick to the surface. Tare the weighing boat or paper. You need to do...volume (e.g. 0.02 g to resuspend in 1 mL), you may place a microcentrifuge tube on the balance and then tare...Pro-Tip* When you weigh out a reagent, you may leave trace amounts of it on the balance. Therefore, it’s best...

...experiment, make sure your workspace is clean and uncluttered. Disinfect your workspace with appropriate materials...Lab coat Close-toed shoes Gloves Eye protection or face shields Eye wash Safety shower Sharps container ...times for each agent. Make sure you have enough space to work at your lab bench. It’s possible that while...or biohazardous materials are splashed onto your face and/or get into your eyes. Wash your hands before...able to close securely. Biohazard signs should be placed in all areas containing biohazardous materials,...

...represents the first decimal place. For the P20, this represents the second decimal place, and for the P2 this...receiving tube, place the pipette tip into the liquid. If this is a new container, place the tip near the...this represents the third decimal place. Procedure How to Pipette Now that you know the different parts ...indicate that the tip is not on the pipette properly. Place the pipette tip into the container you are using...

...solution. Place a mark on the side of the column where the compacted resin is slanted upward. Place column...Desalting Column’s bottom closure and loosen cap. Place column in a 50 mL conical collection tube. Centrifuge...discarding buffer from the collection tube each time. Place column in a new 50 mL LoBind collection tube. Slowly...the reservoir of the column. Fill the remaining space in the column with PBS and pipette several times...through into a waste container. Fill the remaining space in the column with PBS and pipette several times...

...will need to join to create your final plasmid. Adjacent segments should have identical sequences on the... that contain a 5′ end that is identical to an adjacent segment and a 3′ end that anneals to the target...60 bp long, with 30 bp matching the end of the adjacent fragment and 30 bp annealing to the target sequence...one part has half of the antibiotic gene and the adjacent part has the other half. Any colonies should have... antibiotic gene. This trick can also enable replacement of "inverse PCR" reactions with a two-part Gibson...

...Timeline Day 0: Seed and Transduce Cells Day 2: Replace media with fresh media containing selection reagent...an untransduced well by using 150 μL of media in place of virus. Seed 1,000 cells into each well of a 6...directly on top of the virus. Swirl gently to mix and place the dish in the incubator. Incubate for 48–72 h....step can be reduced to 8 h and the media can be replaced with DMEM complete. However, antibiotic-containing... Every 3–4 days, gently aspirate the media and replace it with fresh DMEM complete containing the appropriate...

... coat Close-toed shoes Gloves Eye protection and face shields, as needed Guidelines Your lab coat should...remain in the laboratory. When traveling between lab spaces, take your glove off of one hand and use your bare... on the type of sleeve. Lastly, don’t touch your face! Close-toed shoes and long pants should be worn ...your feet. Eye protection such as goggles and/or face shields can be used as needed. For BSL-2 work always...

...selection curve below. Carbenicillin is used here in place of ampicillin because carbenicillin is more stable... on top of the agar and gently spread over the surface until the liquid is mostly absorbed (there is a...visible volume of pooled liquid remaining on the surface). We use the micropipette tip itself to do the ...suspension onto the agar and gently spread over the surface until the liquid is mostly absorbed. The spreading...

...two methods: Method 1. Place the mixed oligos in a 1.5mL microfuge tube. Place tube in 90-95°C hot block...temperature (~45 minutes). Method 2. Place mixed oligos in a PCR tube. Place tube in a thermocycler programmed...

... opens in a new window) (3,180 cm 2 - the same surface area as 21 x T-175 flasks). Cell stacks provide...these cells are very loosely attached to the dish surface and should be handled carefully. Avoid touching...other than PES. AAV particles stick to many other surfaces, but do not stick to PES. Using a PES filter will... toward the cap. If the media touches the cap, replace it with a new one before putting the CS5 in the...

...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...