...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. ...

...Protocol Video Watch the protocol video below to learn how to perform gel electrophoresis. Equipment Casting...doing diagnostic digests and how to interpret them please see the Diagnostic Digest page. Purifying DNA from...

...Protocol Video Watch the protocol video below to learn how to perform Polymerase Chain Reaction (PCR). ...check out our protocol on plasmid cloning by PCR to learn how to design primers for cloning purposes. What...

...similar outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted...conical tubes, VWR 89039-656 96-well polyester (clear) microplate seal, Thermo Scientific 5701 Microcentrifuge...

...Selective pressure on this heterogeneous cell pool could lead to reduced transgene expression over time, as the...individual cells, which can be difficult to find. Leave the cells undisturbed in an incubator for 7–14 days...

...Protocol Video Watch the protocol video below to learn how to create bacterial glycerol stocks. Procedure...

...reuse or repurpose this SOP in another location, please note that you do so at your own risk; you should...

...as plastic will block much of the UV and with a clean, sterile razor blade, slice the desired DNA fragment...

...bacteria. Video Watch the protocol video below to learn how to inoculate bacteria in liquid culture. Protocol...

...similar outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted...

...disturb the colonies. Count the colonies for at least 2 of the dilutions. Pro-Tip The higher dilution ...

...therefore it is important that the digest go at least 4 hours and as long as overnight. If you are going...

...cell pool, the antibiotic selection should last at least as long as it takes the control (untransduced) cells...