...fresh media containing selection reagent Day 3–14: Change media as needed Day 14–18: Expand and harvest stable...cell line. To do this, treat target cells with a range of doses of antibiotic and determine the lowest ...lentiviral aliquot on ice prior to use. Prepare a range of dilutions of the lentivirus in DMEM complete ...titered your virus beforehand, you can narrow this range according to the results of your titration. Mix ...lentivirus, above) are dying. Perform regular media changes and monitor the growth of the cells. Pro-Tips Depending...culture, so it is important to do regular media changes and maintain optimal growth conditions for the ...absence of cell death, the cell media should be changed every 2–3 days to maintain the dose of antibiotic...

...Option 2 with a buffer exchange/concentration using an Amicon 30 kDa MWCO buffer exchange column. (Optional... Day 1: Purify antibody Day 2 or later: Buffer exchange Equipment Class II, Type A2 Biological Safety ...above 1.0 mg/mL proceed to Option 1 with a buffer exchange using a Zeba Spin 7 kDa MWCO desalting column....the same recombinant antibody. Section 2: Buffer exchange Choose Option 1 or Option 2 based on the concentration...concentration of the pooled sample above. Option 1: Buffer exchange using a desalting column The 10 mL Zeba Spin Desalting...sterile sodium azide to 1 mM. Option 2: Buffer exchange and concentration with an Amicon column Apply ...least 4 additional times to ensure a full buffer exchange. Discard the flow through into a waste container...

...sequences from step B.1 into the oligos below. Do not change the ends; these bases are important for cloning...CO 2 for 12-15 hours. Day 3: j. In the morning, change the media to remove the transfection reagent. Replace...Lentiviral particles can efficiently infect a broad range of cell types, including both dividing and non-dividing...to cells. Days 3+: e. Examine cells each day and change to fresh puromycin-containing media every other...Target cells should be approximately 70% confluent. Change to fresh culture media containing 8 μg/mL polybrene...particles per cell. Addgene recommends that you test a range of MOIs to determine the optimal MOI for infection...Incubate cells at 37°C, 5% CO 2 overnight. Day 3: e. Change to fresh media 24 hours after infection. TIP: If...

...determine when the desired color change has occurred. The color will change from yellow to blue and generally...experimental conditions. When the desired color change has occurred, gently remove the plate seal. Using...generate a standard curve . If you don’t see any color change or dose response, your antibody concentration may...case, use lower antibody concentrations. The ideal range of the standard curve will vary between targets ...your unknown sample’s absorbance falls above the range of the standard curve you will need to either include...

...transformation/replication process. LIC employs long overhangs to form a stable association between fragments...exonuclease functions, T4 DNA polymerase can create overhangs of varying length (typically 10-12 bp) based on...recognition sequence. This can create multiple distinct overhangs with a single enzyme, and remove the restriction...in subsequent reactions. Step 3: Create Vector Overhangs Treat the linearized vector with T4 DNA polymerase...accomplished by gel purification . Step 5: Create Insert Overhangs Treat your purified PCR product with T4 Pol in...

...ensures the accuracy of your experiment and any changes to the amount you are dispensing can negatively...adjustment ring right below the plunger. This ring changes the pipette volume. Below that is the tip ejector...boxes that the tips come in often indicate a volume range that the tip can hold. This should give you an idea...without cross contamination as long as the tip is changed between samples. Tips can come loose in a bag, ...accurately and precisely is important since even small changes in the volume could affect your experiments. Beyond...

...outlined in this protocol is based on an LV titer range of 1E+05–1E+09 TU/mL, where TU is transducing units... This protocol was modified from the publication Wang et al. (2018) . Before Starting Thaw the master ...directly in the center of the well and tilt to a 45° angle. Count to 20 while slowly and gently pipetting the... of the well and tilt the pipette tips at a 45° angle. Count to 20 while slowly and gently dispensing ...Table: Example dilutions and titrations Reference Wang Y, Bergelson S, Feschenko M. Determination of Lentiviral...

... or Klenow DNA Polymerase I for 3′ overhang removal and 5′ overhang fill-in. If you are using blunt ends...amount of DNA in the reaction, incubation time can range from 45 mins to overnight. For diagnostic digests...compatible sticky ends, you will need to fill in the overhangs and conduct a blunt end ligation. Use T4 DNA Polymerase...

...fresh media containing selection reagent Days 3–14: Change media as needed Days 14–18: Stain cells and count...empirically determined. Treat the target cells with a range of doses of antibiotic. Determine the minimum concentration...presence of selective reagent for 12 days with media exchanges every 3–4 days. Colonies were then stained with...presence of selective reagent for 12 days with media exchanges every 3–4 days. After 12 days of selection, no...

... Nonetheless, we recommend performing a buffer exchange before using the purified AAV in vivo . The iodixanol... Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note: Both steps could be completed...Pool clean fractions. Concentration and buffer exchange: Stocks of Pluronic F68 Pluronic F68, 10% solution...adapted from Strobel Benjamin, Miller Felix D., Rist Wolfgang, and Lamla Thorsten. Human Gene Therapy Methods...

...enzyme, or enzymes that have compatible overhangs or no overhangs after digestion, you will need to use ...chance for mis-priming, so you can use a pretty wide range of annealing temperatures, but you may need to increase...cloning. DNA replication by PCR has error rates that range from roughly 1 per 500bp to roughly 1 per 10 million...

...outlined in this protocol are based on an AAV titer range of 5E+12–5E+13 GC/mL, where GC is genome copies ...slowly to avoid generating aerosols. Be sure to change tips each time. Dilution 1 (20X): 5 µL in 95 µL...directly in the center of the well and tilt to a 45° angle. Count to 20 while slowly and gently aspirating ... of the well and tilt the pipette tips at a 45° angle. Count to 20 while slowly and gently dispensing ...

... a short stretch of DNA to a plasmid, such as: Changing a Multiple Cloning Site (MCS) Adding short tags...once annealed can be cloned directly into the overhangs generated by restriction digest of existing sites...to include additional bases to complement the overhangs generated when digesting the vector with EcoRI...

... tubes. Depending on the centrifuge their speed ranges can vary, but are typically lower than some other...unbalanced state can damage the centrifuge and be dangerous for the user. If you do not have a balanced number...are not the same units and are not directly interchangeable. If you need to convert between the two, use...

... a single stranded overhang on the digested end of the DNA fragment. The overhangs, called "sticky ends...sticky ends are compatible, meaning that the overhanging base pairs on the vector and insert are complementary...

...two-part Gibson reaction if you're only making a small change in a plasmid (such as point mutations). Generate...T5 Exonuclease - creates single-strand DNA 3’ overhangs by chewing back from the DNA 5’ end. Complementary...England Biolabs by TelesisBio. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO. (2009). ...

...enzyme, or enzymes that have compatible overhangs or no overhangs after digestion, you will need to use ...only one enzyme or used enzymes with compatible overhangs you will need to verify the orientation of your...

...red were calculated using this plasmid, but will change if you use a different plasmid. Sample Calculation...dilutions 5–8 Note: at Addgene, samples typically range from 1 x 10 12 GC/mL to >2 x 10 13 GC/mL and we ...) ~ 1.0, E (efficiency of PCR) ~100% (90%–110% range is acceptable) Baseline removal: all samples will...

...drop down menu and select Scatter chart. Change the data range and select the cells where the x-axis is...Open . Choose the location of the file to open. Change the image type to 8-bit . Select Image . Select...

...the medium is acidified (i.e., the medium appears orange/yellow if using a standard phenol red pH indicator...control. 1 lentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid #52962 ) and is described in Improved...libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8):783-4. (Link opens...