... A diagnostic restriction enzyme digest takes advantage of the fact that restriction enzymes cleave DNA...form of diagnostic digest is one in which you just want to verify that the plasmid that you have is the ...the correct orientation. In the example below we want to know how to differentiate between two clones...or enzymes) that cuts within the insert, but significantly off center (ideally around 1/3 of the way from...

... As the name suggests, light microscopes take advantage of the physical properties of light to detect ...detect small objects. Two of the most important properties of a microscope are magnification (the ability to...see several pieces of information, but the most important (for now) is the magnification power, such as ...your images in the future. Conclusion Like any important laboratory instrument, you should be sure to take...

...been sufficiently selected with the appropriate antibiotic so that every cell in the culture has, in theory...maintain cells in selective medium at a reduced antibiotic concentration (e.g., half the concentration that...Sample Calculation First, use a hemocytometer to quantitate the homogenized cell solution. Homogenized cell... additional 3 weeks under blasticidin selection. Anti-Cas9 Western blotting was performed on whole-cell...

...Centrifuge for 5 min at 100 x g . Gently aspirate supernatant. Wash cell pellets in 500 µL PBS. Centrifuge ...Centrifuge for 5 min at 100 x g . Gently aspirate supernatant. Extract genomic DNA according to the GeneJet Genomic...transfer the plate to the Droplet Reader. Open the QuantaSoft software to set up a new plate layout. Designate...the dilutions. RPP30 copies should be relatively constant across samples. In the RRE example below, 2-fold...

... in healthy humans. Many of the precautions are meant to protect individuals from potential accidents ...don’t touch your face! Close-toed shoes and long pants should be worn to protect your legs and feet from...the safest way possible. Protecting yourself is important! Disclaimer: The SOP presented here has been designed...

...Introduction Restriction enzyme digestion takes advantage of naturally occurring enzymes that cleave DNA...given digestion will depend on the amount of DNA you want to cut. By definition: one unit of enzyme will cut...Dam or Dcm methylase positive strains will be resistant to cleavage at certain restriction sites. See ...

...harvesting. Filter supernatant through a 0.45 μm PES filter. The viral supernatant can be stored at 4 ...at 4 °C between harvest. Centrifuge the viral supernatant at 2100 rcf for 5 minutes to pellet any packaging...

...measurement in the next step. The second stop is meant to expel excess liquid from the tip once you’ve ...Conclusion Pipetting accurately and precisely is important since even small changes in the volume could affect...Beyond knowing the proper way to pipette, it’s important for scientists to calibrate their pipettes to ...

...Note: If you add EtBr to your gel, you will also want to add it to the running buffer when you run the...separate larger bands. 10% Rule For each sample you want to load on a gel, make 10% more volume than needed...microliters can be lost in pipetting. For example, if you want to load 1.0 μg in 10 μL, make 1.1 μg in 11 μL. Reference...

...droplet digital PCR (ddPCR) to titer purified recombinant Adeno-associated viral vectors (AAV). This protocol...ddPCR, read this blog post on ddPCR for AAV quantification. This protocol was modified from the publication...transfer the plate to the Droplet Reader. Open the QuantaSoft software to set up a new plate layout. Designate...

...relatively limited MCS (BamHI - EcoRI - SalI) and you want to expand that with the addition of NdeI, PacI, ...annealed oligos with 45μL nuclease-free water and quantify the concentration (should be about 8ng/μl). Mix...

... Digest Introduction Sequence verification of important plasmid features (such as the gene/insert, fusion...you sequence any portion of the plasmid that is important for your experiments. There are a number of factors...

... or paper. You need to do this because you don’t want to include the weight of the weighing boat or paper...Conclusion Appropriately weighing reagents is important to ensure the accurate composition of your solutions...

...Over-Agar Antibiotic Plating Create plates to culture bacteria in the lab Over-Agar Antibiotic Plating ...

... It is not recommended to subject lentiviral supernatants to multiple freeze-thaw cycles. Procedure Seed...titer lentiviral vectors (10 5 TU/mL). If you anticipate that your viral stock will be higher titer, consider...

...located before you start. Use the eyewash station if unwanted or biohazardous materials are splashed onto your...Protecting yourself and working smart in the lab is important! Disclaimer: The SOP presented here has been designed...

...a T75 flask in a volume of 15 mL. Do not add antibiotics to the media. Use cells that are below passage...between batches of PEI depending on the user, quantities of chemical used, volumes, pH adjustment etc....

...became automated with the discovery of a heat resistant DNA polymerase from the thermophilic bacterium...are complementary to the 3’ ends of the sense and anti-sense strands of the template DNA. They are strands...

...toward one another. The size of the primer is very important as well. Short primers are mainly used for amplifying...

...provided by your polymerase manufacturer. It is very important to remove all free nucleotides from your PCR product...