...Preparation 1 M NaCl/PBS-MK buffer Dissolve 5.84 g of NaCl, 26.3 mg of MgCl 2 and 14.91 mg of KCl in 1× PBS ...gradient for AAV purification. Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note...Dissolve 26.3 mg of MgCl 2 , and 14.91 mg of KCl in 1× PBS in a final volume of 100 mL. Sterilize by passing... step: mix 4.5 mL of 60% iodixanol and 13.5 mL of 1 M NaCl/PBS-MK buffer 25% iodixanol step: mix 5 mL ...Hamilton needle, taking care to avoid bubbles (Figure 1). 8 mL of 15% iodixanol step 5 mL of 25% iodixanol...disturb the gradient!** Collect Fractions Option #1 Prepare a row of roughly 20 open 1.5 mL microcentrifuge...with a 16 ga needle and start collecting 0.5 mL to 1 mL fractions per tube. Avoid the proteinaceous material...

...June 14, 2023 Workflow Timeline Day 1: Run SDS-PAGE and stain gel Day 1 or later: Image analysis Video Watch...incubate for 1 h with gentle agitation on a rocking platform. Pour off the water in the sink. Figure 1 Add 100...lane Example for AR0018 (lane 2 in Figure 1): Sample Peak 1 (contaminant) Peak 2 (contaminant) Peak 3 ...antibodies using Coomassie stain. Equipment Heat block 1–10 µL single channel pipette 2–20 µL single channel...deionized water. Gently invert to mix. Procedure Section 1: SDS-PAGE Add 5 µL of 4X sample buffer to each sample...attach to a power supply. Run the gel at 150 V for 1 h . *Pro-Tip* If the samples are running unevenly ...Add 20 mL of SimplyBlue SafeStain and incubate for 1 h with gentle agitation on a rocking platform. Pour...

...annealing temperature step-wise by 1-2°C. The rate of DNA synthesis is ~1-2 kb/min. The extension time can...now bind to the primer DNA sequence. Extend DNA for 1 minute at 72°C: The Taq polymerase has an optimal ...DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 mM each nt) 2.5 μL Forward Primer ...primer melting temperature (Tm). Set extension step at 1-2 minutes per kilobase of product depending on whether...working concentration of each primer (10uM) by making a 1:10 dilution of the stock. For example, add 100µl of...Gel Procedure Primer Design and PCR Design Primers. See our protocol on how to design primers . Note: Primer3...

... dilution Dilution 1 (DNase step) 5uL AAV stock 45 uL 10x 10x Dilution 2 5uL Dil. 1 95 uL 20x 200x Dilution...The reference material should have a titer within 1-log of the expected titer of the samples being tested...Amount for ONE Reaction Amount for 100 reactions (1 x 96 well plate) Unversal SYBR Master Mix 2X 10 μL...make a small aliquot of each standard (enough for 1 or 2 plates) and store at -20°C. Once a standard is...not freeze it again but store at 4°C and use within 1 week. Keep track of the Ct value for each standard...standard curve plasmid (2x10 9 stock made in step #1): Volume of 2x10 9 stock or previous dilution (μL).../mL. We recommend always using a reference within 1-log of the expected titer. *Pro-Tips* The quality ...

...Last Update: January 20, 2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells Equipment...into 5 mL PBS. Protect from light. Procedure Section 1: Seeding cells Place a sterile poly-D-lysine coated...concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL of the diluted antibody to the...concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL fluorescently-labeled secondary...with a laboratory wipe to remove excess liquid. Add 1 drop of anti-fade mounting medium to the microscope...This protocol describes the basic steps for fixing and staining cells in culture with a primary antibody...antibodies to detect antigens in cells. Here we describe the basic steps for fixing and labeling cells ...

... resistance) Procedure Day 1 Prepare carbenicillin to a concentration of 1 mg/mL – 4 mg/mL in LB medium...lawn of E. coli and no apparent selection. 150 µL of 1 mg/mL Carbenicillin plated over-agar Plate shows several...over-agar Plate shows less individual colonies than the 1 mg/mL plate and effective selection. 150 µL of 4 mg...several individual colonies with smaller size than the 1 mg/mL and 2 mg/mL plates and effective selection. ... Bacterial Culture Introduction This protocol describes methodology for plating antibiotic over-agar for...

...get more colonies if you use 1 μl of a 1:5 or 1:10 dilution rather than 1 μl directly....and then (optional) incubate in 37°C incubator. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 20-50 ...shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30...Because of this, nearly all plasmids (even those designed for mammalian cell expression) carry both a bacterial...

...situations where the 3:1 ratio is not working or when doing more complicated cloning. While 3:1 will get you in...the insert is smaller than the vector) a 3 insert : 1 vector molar ratio will work just fine. We recommend... Optimizing the Vector:Insert Ratio: Although a 3:1 insert to vector ratio is usually sufficient, you ...phosphodiester linkages, which permanently join the nucleotides together. After ligation, the insert DNA is physically... likely to ligate to itself rather than to the desired insert. If you are in this situation, it is important...Note: Because ligase buffer contains ATP, which degrades upon freeze/thaw cycles, it is a good idea to ...

...10 mg/mL) Procedure Pouring a Standard 1% Agarose Gel: Measure 1 g of agarose. *Pro-Tip* Agarose gels are.... Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and...different buffers and do not use water). Microwave for 1-3 min until the agarose is completely dissolved (but...the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and ...be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip. Place newly...Always Run to Red. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove...-30 mins, replace EtBr solution with water and destain for 5 mins. Using any device that has UV light,...

... chloride 12.0 g agar-agar 1 L Sterile H 2 O Sterile plates of your desired size - we usually use 60 mm...bottle for autoclaving. We make 400 mL of agar in 1 L bottles and 200 mL of agar in 500 mL bottles. The...measure out 100 mg of ampicillin powder, add it to 1 mL of water, dissolve by vortexing, and filter sterilize... You should not store your plates for longer than 1 month at any temperature and should always check for... for contamination prior to use. Negative Result 1: Both Strains Grow Assuming the appropriate strains...next to the flame and begin pouring. Measure your desired amount of agar with a pipete for the first plate...

...has error rates that range from roughly 1 per 500bp to roughly 1 per 10 million bp depending on the polymerase...recipient plasmid to insert ratio of approximately 1:3. Since the number of base pairs for each varies,...cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent cells...plasmid of interest. For this example, we will describe how to copy a cDNA from one vector into a new ...(YGOI) for ligation into a recipient plasmid. Designing primers for PCR based cloning: The basic PCR primers...that: Do not cut within your insert Are in the desired location in your recipient plasmid (usually in ...examine the DNA sequence that we want to amplify and design primers that will bind to and replicate it. The...

...annealing can be achieved by one of two methods: Method 1. Place the mixed oligos in a 1.5mL microfuge tube....vector in molar ratios (vector:insert) between 4:3 and 1:6 in a standard ligation reaction (ex. to ligate an...oligo overlap cloning, you can design a set of oligos containing your desired restriction sites and add them...that will pay off for years to come. Design Briefly, we will design overlapping oligos that once annealed...procedure will simply differ in terms of primer design). Let's assume that your favorite vector has a ...digest of existing sites in the original vector. Designing oligos: To add NdeI, PacI, AscI and MfeI sites...between the EcoRI and SalI sites of the vector, we design a top oligo with each of the additional sites in...

...dispense small amounts of liquid (think: 0.1 µL to 1 mL). When working in a laboratory, properly dispensing...right. Pipette Dispense Volume P2 0.2 to 2 µL P10 1 to 10 µL P20 2 to 20 µL P100 10 to 100 µL P200 20 ...and the third (red) digit is hundredths. Therefore, 1 µL would read as 100 (as shown in the picture above...maximum possible setting on the pipette, set the desired volume of the liquid on the pipette. Depending ...of the pipette. Select the pipette tip that is designed to fit your pipette. To load a tip onto a pipette...from the container making sure not to touch the sides of the container with the pipette. Discard the pipette...

...Figure 1 depicts an image of a compound light microscope with the main components labeled: Figure 1: Diagram...magnifies it 10 times, and so on. The ocular lens also provides magnification and the power should be provided...provided on the microscope; often this lens provides 10x magnification. To determine the final magnification...that object! Equipment Light Microscope Prepared Slides (or other sample that can fit on a microscope stage...eyepiece so that you can see your image through both sides. Once you are happy with the lighting, use the coarse...

...recipient plasmid to insert ratio of approximately 1:3. Since the number of base pairs for each varies,...cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent cells...restriction enzyme digest (subcloning), including design and experimental procedures. Protocols...easily move YGOI into a mammalian expression vector. Design (Choosing enzymes) Many DNA analysis tools, including..., but do not cut within your insert Are in the desired location in your recipient plasmid (usually in ...your recipient plasmid as well as a specifically designed test digest later to verify that the insert was... fear. You have other options, such as: Adding desired restriction sites to flank your insert : You can...

... measure your samples, place 1-2µL of mini-prepped DNA onto the pedestal. Close the lid and click measure...Repeat for each sample. Note: Keep in mind that despite the accuracy of the NanoDrop, if two consecutive...

...quicker. *Pro-Tip* Set up the water bath 30 minutes to 1 hour before you need to use it to allow the temperature... of bacteria or fungi. There are disinfectants designed to be used in water baths with instructions on...bath on and set the appropriate temperature as described in your protocol. How to set the temperature will...Set up your water bath so that it will reach the desired temperature by the time you need it for your experiment...water bath to prevent evaporation and maintain the desired temperature. This also helps the water bath heat...water bath even if you are using disinfectants as described above in step 3. You will also need to maintain...

...ssDNA. (Rabe & Cepko, 2020). Incubate the mix for 1 hour at 50 °C or follow manufacturer's instructions... Procedure Design your plasmid and order primers (see figure to the right). When designing your plasmid...and enrich for correctly assembled plasmids by designing primers to split an antibiotic resistance gene...and yield. If there are significant amounts of undesired product, gel-purify DNA segments. Otherwise, ... High-Fidelity DNA Polymerase - incorporates nucleotides to “fill in” the gaps in the annealed DNA fragments...<150 bp). *Pro-Tip* Please note that the way to design the “stitching” primers and the amounts of primers...window) . NEB has other resources, such as a primer design tool (Link opens in a new window) . Gibson Assembly...

...solution, add 2-2.5 volumes 95% or 100% ethanol and 1/10 volume of 3 M Na-acetate (pH 4.8). Invert the microfuge...Last Update: Feb. 8, 2018 Equipment Desktop microcentrifuge Desktop vortexer Vacuum (optional) Reagents... several μg/μL. The protocol below is meant to describe the general procedure for purifying plasmid DNA...