... to titer lentiviral preparations that confer antibiotic resistance.... to titer lentivirus preparations that confer antibiotic resistance. Determining the titer of your lentiviral...selection markers. Note this assay requires staining resistant colonies with crystal violet solution and, therefore...media as needed Days 14–18: Stain cells and count resistant colonies Equipment Class II, Type A2 Biological...lines. It is not recommended that lentiviral supernatants be subjected to multiple freeze-thaw cycles....beginning a colony formation assay, the dose of antibiotic required to kill your target cell line needs ...Treat the target cells with a range of doses of antibiotic. Determine the minimum concentration (Link opens...

...Information The final step in the construction of a recombinant plasmid is connecting the insert DNA (gene or...desired insert. If you are in this situation, it is important to treat the digested vector backbone with a phosphatase...setting up the ligation reaction itself, it is important to determine the amount of cut insert and vector...in a new window) ligation calculator to easily quantify how much vector and insert DNA to use. Combine...it will have some amount of background. What you want to see is that your vector + insert ligation has...viability of competent cells and verifies the antibiotic resistance of the plasmid Cut vector - Background...average size genes and vectors, this ratio is really meant to refer to the molarity of DNA ends available for...

...organelles, lipoproteins, and macromolecules. Importantly, iodixanol is inert and non-toxic to mammalian...protocol is composed of steps that separate out contaminants from an impure AAV preparation. The 15% iodixanol...macromolecules. The 40% and 25% steps are used to remove contaminants with lower densities, including empty capsids...iodixanol step Carefully add up to 5 mL of clarified supernatant on top of the gradient. Use 1X PBS (or formulation... to collect the solution, or you will lose a significant amount of your virus. When the first fraction.... Image adapted from Zolotukhin, S., et al. "Recombinant adeno-associated virus purification using novel...Left image adapted from Zolotukhin, S., et al. "Recombinant adeno-associated virus purification using novel...

...homology region. Hairpins in this region can significantly reduce the efficiency of two homologous ends...assembled plasmids by designing primers to split an antibiotic resistance gene to effectively create an extra...extra part, one part has half of the antibiotic gene and the adjacent part has the other half. Any colonies... should have at least the correctly assembled antibiotic gene. This trick can also enable replacement ...gel to check for size and yield. If there are significant amounts of undesired product, gel-purify DNA ... raw PCR mix can work fine in an assembly if you want to save time. Working on ice, combine segments in... product by restriction digest . Sequence the important regions of your final plasmid, particularly the...

... tub. Disinfectant may be added to prevent growth of bacteria or fungi. There are disinfectants designed...Bath 10% bleach or 70% ethanol Distilled water Disinfectant Thermometer Water bath weights and floats Reagents...allow the temperature to stabilize. Use water resistant markers, such as permanent markers, to label your...water bath, be sure to use a marker that is also resistant to chemicals such as ethanol. Writing on the tops...clean out the water bath even if you are using disinfectants as described above in step 3. You will also ...

...appropriate antibiotic) Bacterial stab Procedure Obtain an LB agar plate with appropriate antibiotic. Label...add the antibiotic resistance and your initials. Labeling within a laboratory setting is important for organization...and need to purify plasmid DNA from it, you will want to isolate an individual clonal population (single...

... save the pellet, the supernatant, or both. Conclusion Centrifuges are important tools for many different...volume as your samples. If your samples are significantly higher density than water, then you may need... The centrifuge should come up to speed with a pleasant hum. If you hear loud noises or the centrifuge...sample while the liquid portion is known as the supernatant. Depending on your experiment, you may be required...

...on ice and transfer supernatant to a sterile 500 mL bottle. Process the supernatant as follows: Filter ... Add 25 mL of PEG solution to each 100 mL of supernatant. Split into 2 x 500 mL sterile bottles as needed... at 3900 rpm for 15 min at 4 °C. Discard the supernatant and resuspend the pellets in a total of 5 mL ...now ready to purify your prep . The clarified supernatant can be kept overnight at 4 °C before proceeding...

...Background Information Bacterial glycerol stocks are important for long-term storage of plasmids. Although you...glycerol stocks of their plasmids. This way, when you want to make more plasmid DNA, the plasmid will already...be stored at 4°C for a few weeks. However, if you want to store bacteria for a longer time, you will need...completely and will improve the shelf life. It is very important that you shake the glycerol before freezing (5...

... 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...

...Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived...