...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...

...Learn about selecting and using a centrifuge to separate different components in a liquid sample....for your samples, ensure that the centrifuge is clean and that everything appears to be working smoothly... The centrifuge should come up to speed with a pleasant hum. If you hear loud noises or the centrifuge...samples, or you, so be sure to keep your centrifuge clean and balanced....

...Learn how to transform E. coli with your plasmid of interest....efficiency. The lowest efficiency cells (usually the least expensive) are fine for transforming plasmid DNA...Protocol Video Watch the protocol video below to learn how to isolate single bacterial colonies. Equipment...antibiotics it is a good idea to outgrow for at least 20-30 mins) I get very few if any transformants ...

...similar outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted...Protocol Video Watch this instructional video to learn how to affinity purify recombinant antibodies. Workflow...Starting Wipe down all pipettes and equipment with 10% bleach prior to use. Warm the affinity column, binding...through into a waste container. Repeat steps 29-31 at least 4 additional times to ensure a full buffer exchange...

...similar outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted...antibody Video Watch this instructional video to learn how to use Western blotting to visualize a protein... 14,000 x g at 4 °C . Transfer supernatant to a clean microcentrifuge tube. Use the lysate immediately...reagent for 5 min at RT . Cover the membrane with clear plastic and use a gel imager with chemiluminescence...

...slowly add hydrochloric acid until the solution clears. Check the pH of the solution and use hydrochloric...cell pellets and sonicate 5 x 1 sec pulses with at least 5 minutes on ice between each pulse, 50% amplitude...centrifugation at 3900 rpm for 10 min at 4 °C. Transfer the cleared lysate to the tube containing the resuspended virus... ~18 mL from steps 12 & 13). Benzonase is an endonuclease that will degrade any residual DNA carried over...

...Learn how to streak bacteria on an LB agar plate to obtain single colonies....Protocol Video Watch the protocol video below to learn how to isolate single bacterial colonies. Equipment... only the first) in each new section crosses at least one line of the previous section so that it will...

...AATTAATT CCGCGCGG GTTAAC CAGCT - 5' Note: We could leave off the 3’ G on each oligo (and the complementary...microfuge tube. Place tube in 90-95°C hot block and leave for 3-5 minutes. Remove the hot block from the heat...Ligation Dilute 5μL of annealed oligos with 45μL nuclease-free water and quantify the concentration (should...

...Restriction enzymes are naturally occurring bacterial endonucleases that recognize a large range of DNA sequences... advantage of the fact that restriction enzymes cleave DNA at specific sequences called restrictions sites...Protocol Video Watch the protocol video below to learn how to analyze a restriction digest. Equipment Electrophoresis...

...In this protocol you will learn the basics of how to use a light microscope....entire courses on microscopy and still have more to learn, so, for this protocol, we'll focus on one of the...easily impair microscope performance. Be sure to clean up your work area and microscope after use and store...

...basic PCR primers for molecular cloning consist of: Leader Sequence: Extra base pairs on the 5' end of the... existing software (a quick internet search will lead you to (Link opens in a new window) here and many...therefore it is important that the digest goes at least 4 hours and as long as overnight. If you are going...

...should be added upstream in order for the enzyme to cleave efficiently (e.g. GCGGCG-restriction site-your ...Protocol Video Watch the protocol video below to learn how to design primers for PCR. Reference Page Top...

...contact Addgene about the accuracy of your plasmid, please email [email protected] and include: The sequencing...trace file. If the mutation is not an artifact, please email [email protected] with the sequence, trace...

...Lentivirus is generally considered biosafety level 2+. Please ensure that you are in compliance with your institution...counting cells with multiple integration events leading to underestimation of the true titer. Calculate...

...slowly add hydrochloric acid until the solution clears. Check the pH of the solution Use hydrochloric ... the plasmid should also be propagated in an endonuclease negative E. coli strain such as NEB stable. ...

...slowly add hydrochloric acid until the solution clears. Check the pH of the solution. Use hydrochloric...the plasmids should also be propagated in an endonuclease negative E. coli strain such as NEB stable. ...