... 37 °C for 5 min. Note: Ribonuclease A (RNase A) is a pancreatic ribonuclease that digests single-stranded...colonies and to generate liquid bacterial cultures , please see those pages. Several companies, such as (Link... Note: This step causes the bacteria to lyse, releasing their contents, including plasmid DNA, into solution...proteins and genomic DNA to precipitate, while leaving the smaller plasmids free in solution. Pellet the...times to mix the contents. The contents will become clear and thicker as the proteins and DNA are denatured...temperature on the highest setting. Note: You should see clearly separated layers: Top Phase - Aqueous DNA phase...precipitate. Centrifuge solution at high speed (at least 12,000 rpm) for 15-30 min at 4°C. Notes: Pellet ...

...setting that gets the sample to at least 121 ℃ under 20 psi for at least 30 min. The high pressure will prevent...bath. Pro-Tip You should leave the molten gel-mix in the water bath for at least 5 min. Do not let any of...the autoclave process if your sample has spent at least 10 min at 121 ℃. Use lab tape to label the bottle...initials, the date, and the bottle contents. This will clear up any confusion later if your forget your bottle...where you’ll be pouring your plates - be sure to leave room for your bottle of molten gel mix, a tube rack...opening the door to the autoclave just a crack and leaving it open that way for ~10 min. This will allow any...microwaving (if you microwave, beware of over-boiling!). Leave your plates out on the bench to solidify. Pro-Tip...

...to hazardous materials. Video Watch the video to learn more about safety in the lab. Equipment Lab coat...start your experiment, make sure your workspace is clean and uncluttered. Disinfect your workspace with appropriate...you may use 70% ethanol, quaternary ammonium, or bleach solutions. Ask your lab supervisor what is available...These procedures should be done underneath a hood. Clean any spills immediately, and decontaminate as necessary...necessary. For large spills that cannot be easily cleaned, evacuate and close off the area and alert the ... of the BSL-2 area. BSL-2 laboratories must be clearly marked as “BSL-2.” The names and contact information...information of the laboratory manager should be clearly visible in the room and on the door. BSL-2 centrifugation...

...Liquid waste should be decontaminated with at least 10% bleach. Laboratory materials that come in contact... to follow, please contact your institution’s safety office. For more information, please see Addgene'...cloning vector available to the scientific community. Please cite Moffat et al., Cell 2006 Mar; 124(6):1283-...TRC cloning vector has a 1.9kb stuffer that is released by digestion with AgeI and EcoRI. shRNA oligos...improves transduction efficiency by facilitating nuclear import of the vector’s preintegration complex in...52%. Sense 3’ end should have low stability – at least one A or T between position 15-19. Avoid targeting...’s BLAST program. Select sequences that have at least 3 nucleotide mismatches to all unrelated genes. ...

...should be at least 1000X greater than number of perturbations in library. Example: At least 10 colonies...process my Addgene pooled library? Introduction Please refer to our pooled library material pages for ...basic knowledge of the library being amplified. Please see our protocol on Bacterial Transformation . ...Molecular Devices, X602) Pro-Tip Pour these plates at least one day in advance to allow adequate time to fully.... Prewarm 12 mL recovery media at 37 °C (for at least 15 minutes). Prewarm 3X LB Agar + Antibiotic plates...Electroporation involves the use of high voltages, please use caution when activating pulse and follow all.... Day 2 (morning) Before beginning, prechill at least four conical tubes on ice and ensure access to sterile...

...tip. Before releasing the plunger, lift the pipette tip out of the liquid. Once you are clear of the liquid...multichannel pipette. Please watch Addgene’s protocol on multichannel pipetting to learn more....Learn about selecting the correct pipettor and pipette tip, how to dispense liquids, and how to handle.... Once the end of the tip is submerged, slowly release the plunger, allowing it to go back to its resting... the plunger all the way to the second stop to release the liquid. Pro-Tip Dispense until the first stop...liquid you can slowly release the plunger. Remove the entire pipette from the container making sure not...

...pipet each dilution up and down at least 10 times, and use at least half of the final volume (mix with...standard curve RNase-free DNase 10X DNase buffer Nuclease-free water Microcentrifuge tubes 96-well optical...Always run standards and samples in duplicate at least If possible, include an AAV reference sample of ...0.15 μL 15 μL 100 μM Reverse Primer 0.15 μL 15 μL Nuclease Free Water 4.7 μL 470 μL Procedure Prepare a plasmid...10 9 stock or previous dilution (μL) Volume of nuclease-free water (uL) Molecules per μL 10 90 2 x 10 ...Dilution Series Volume of sample (uL) Volume of nuclease free water (uL) Dilution factor Total dilution...well and mix well by pipetting back and forth at least 5 times. Seal plate with transparent film. Centrifuge...

...without ligation. Because of its dual polymerase/exonuclease functions, T4 DNA polymerase can create overhangs... as using the backbone manufacturer's suggested leader sequence fused to your gene of interest, in frame... which a single dNTP is included to stop the exonuclease function of the enzyme and shift its activity...sequence. 5' and 3' primers will have different leader sequences, but operate on the same principle (homologous...polymerase protocol), causing the enzyme to perform exonuclease activity until the first "G" in the sequence.... the polymerase reaction is favored over the exonuclease reaction, the polymerase will add back the guanosine... proceeding, as they will interfere with the exonuclease activity of T4 Pol in the following step. This...

...takes advantage of naturally occurring enzymes that cleave DNA at specific sequences. There are hundreds of...11th, 2016 Protocol Video Watch the video below to learn how to analyze your restriction digest results. ... is determined under ideal conditions with very clean DNA, so using a little more enzyme is advisable....cloning, it is recommended that you digest for at least 4 hours. If you will be using the digested DNA for...such as a (Link opens in a new window) QIAGEN DNA cleanup kit . See the enzyme manufacturer's instructions...methylase positive strains will be resistant to cleavage at certain restriction sites. See (Link opens ...reasons, including high glycerol concentration. Learn more at (Link opens in a new window) NEB's website...

...with 10% bleach. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that...targets. Primers and a probe against the cellular ribonuclease P/MRP 30 kDa subunit (RPP30) are used as a control...a well of a 6-well plate (1 dilution per well). Leave one well untransduced (add 150 µL of DMEM complete...place on ice. Wipe down a DG8 cartridge holder with bleach and place it in the Biological Safety Cabinet (...primers/probe (HEX/VIC) 1 µL 9 µL 900 nM, 250 nM Nuclease-free water 4 µL 36 µL Total Volume 16 µL 144 µL...wiping down all pipettes and equipment with 10% bleach prior to use and keeping all reagents and samples...Sample Data When analyzing data there should be a clear distinction between negative droplets (black) and...

...use. Wipe down all pipettes and surfaces with 10% bleach. Safety Warnings AAV is generally considered biosafety...require BSL-2 handling depending on the insert. Please ensure that you are in compliance with your institution...place on ice. Wipe down a DG8 cartridge holder with bleach and place in the Biological Safety Cabinet (BSC...just gently cover so nothing falls into the plate. Leave dilutions on the chilled 96-well freezer block in...Reverse ITR Primer (10 µM) 1.8 µL 16.2 µL 900 nM Nuclease-free water 5.9 µL 71.55 µL Total Volume 20 µL ...Sample Data When analyzing data there should be a clear distinction between negative droplets (black) and...wiping down all pipettes and equipment with 10% bleach prior to use and keeping all reagents and samples...

...reagent, you may leave trace amounts of it on the balance. Therefore, it’s best to clean the balance before...In this protocol you will learn the basics of how to weigh reagents. Protocols... of your experiment. In this protocol, you will learn the basics of how to weigh reagents. Wear the appropriate... it. Make sure that your scoopula or spatula is clean, and sterile (if necessary), to prevent any potential...that any spills produced during the protocol are cleaned up. Discard the weighing boat or weighing paper...

...bath cover are cleaned before filling the water bath with water. You may use a 10% bleach solution or a...you can leave the water in it from previous uses. However, you will still need to regularly clean out the...water bath in the lab. Equipment Water Bath 10% bleach or 70% ethanol Distilled water Disinfectant Thermometer... To properly maintain the water bath, empty and clean it regularly. Determine the amount of water needed...

...Microscope Learn about the parts of a light microscope and its use Weighing Reagents Learn how to weigh...you'll find these protocols useful in your own work. Please feel free to email us at [email protected] with ...Protective Equipment (PPE) for BSL-1 and BSL-2 Labs Learn how to best protect yourself when working in BSL... maintenance and use Watch the Video! Pipetting Learn about selecting the correct pipettor and pipette...handle the pipette Watch the Video! Centrifugation Learn about selecting and using a centrifuge to separate...

...ssDNA ends from the ssDNA-specific endonuclease activity of T5 Exonuclease and furthermore reduces secondary...Protocols Summary In 2009 Dr. Daniel Gibson and colleagues at the J. Craig Venter Institute developed a ...has the other half. Any colonies should have at least the correctly assembled antibiotic gene. This trick...specific and unique function for the reaction: T5 Exonuclease - creates single-strand DNA 3’ overhangs by chewing...too short to make its own part (<150 bp). Pro-Tip Please note that the way to design the “stitching” primers...

...Barr nuclear antigen (EBNA). Safety Warnings HEK293 cells are considered biosafety level 2. Please ensure...similar outcomes when using these protocols. However, please be aware that your protocol may need to be adjusted.... Wipe down all pipettes and equipment with 10% bleach prior to use. Reagent Preparation 100 mM Valproic...transfer 0.5 mL of HEK293 cell suspension into a clean microcentrifuge tube. Pro-Tip Cells settle quickly... sampling. Transfer 10 µL of trypan blue into a clean microcentrifuge tube. Vortex the cell suspension...

...purity. Only the cleanest fractions should be kept and pooled for dialysis. Pool clean fractions. Concentration...genome-containing virions. Phenol red is added to clearly distinguish the steps. This method will help enrich...Protocol Video Watch the protocol video below to learn setup and use an iodixanol column gradient for AAV...discard the flow through. Repeat this step as needed. Please note that iodixanol is not easily removed. After...gradient before ultracentrifugation. The steps are clearly distinguishable thanks to the use of phenol red...

...antibody sample to the appropriate well. Pro-Tip Leaving clear lanes between samples will make quantifying ...similar outcomes when using these protocols. However, please be aware that your protocol may need to be adjusted...analysis Video Watch this instructional video to learn how to determine purity and concentration of recombinant...Figure 1 Recombinant antibody preps should have 2 clear bands at ~50 kDa and ~25 kDa corresponding to the...processed correctly in the cell (e.g., failure to cleave the signal peptide) and may not be functional. ...

...protocol material, visit our Addgene Protocols page . Please feel free to email us at [email protected] with ...Ultracentrifugation Protocol Mulitchannel Pipetting Technique Learn how to pipette multiple samples at once using a ...microbiology, molecular biology or cell biology. Learn some of the key strategies of aseptic technique,...including personal protective equipment, setting up a clean workspace, and maintaining sterility while working...installment, we sit down with Senior Scientific Project Leader Eric Perkins to discuss the various positions he...

...Learn how to add antibiotic to a plate after it's already been poured and solidified....Protocol Video Watch the protocol video below to learn how to spread antibiotic over-agar. Equipment Pipette.... Incubate the plate at room temperature for at least 30 minutes with the lid on to give the antibiotic...of Carbenicillin stock used (150 µL per plate). Please note we have found that there is generally a broad...