...primer design software to ensure a melting temperature between 50-60°C for your PCR primers. Step 2: Linearize...20-30 Eluted DNA 10-50 ng/μl 1 dGTP (100mM) 2.5 mM 2 DTT (100 mM) 5 mM 1 BSA (10 μg/μl) 0.25 μg/μl 1 T4...your treated vector and insert at a molar ratio of 1:2 or 1:3, using between 20 and 50 ng of vector per annealing... reaction is now ready for transformation. Use 1-2 μl of annealing reaction for each transformation, ...experimental design. Search Addgene's collection of empty LIC cloning vectors Protocol Step 1: Design Your Primers... Primers Primer design for LIC is often as simple as using the backbone manufacturer's suggested leader...nucleotide dGTP in the reaction (exclude all other nucleotides from standard polymerase protocol), causing the...

...Workflow Timeline Day 0: Seed and Transduce Cells Day 2: Replace media with fresh media containing selection...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...the appropriate antibiotic. Incubate the cells for ~2 weeks. All of the cells in the untransduced (negative...disturb the colonies. Count the colonies for at least 2 of the dilutions. *Pro-Tip* The higher dilution wells...stained with 0.1% crystal violet and counted. Figure 2: A549 cells were transduced with the indicated serial...for Lentivirus You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service...

...Day 1: Seed individual cells in a 96-well plate Day 2–14: Monitor cells for growth and expand cells Day ...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...channel pipette 200–1000 µL single channel pipette CO 2 incubator Pipet controller Hazardous waste container...approximately 50–60% confluent. For 293T cells this is about 2 × 10 6 cells in a 10 cm dish. Each 10 cm dish should...the highest or lowest transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal ...inferred by the differences in colony size. Figure 2: Cas9 expression in monoclonal cell lines generated...Vector Guides Virus Blog Posts Addgene Protocols Viral Service Introduction This protocol describes how ...

...agarose concentrations (e.g., 2 g of agarose in 100 mL of TAE will make a 2% gel). Mix agarose powder with...gels are commonly used in concentrations of 0.7% to 2% depending on the size of bands needed to be separated...concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds...allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases...be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip. Place newly...Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and ...Always Run to Red. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove...

...the HeLa cells to grow to the desired density before labeling. Section 2: Fixing and permeabilizing cells...Fluorescent microscope 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... plate 4% Paraformaldehyde 5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) Bovine serum albumin (BSA) Triton...of the diluted antibody to the wells and incubate 2 h at RT . Remove the primary antibody and dispose ...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 ... container. Dilute the primary antibody to the desired concentration in antibody dilution buffer. *Pro-Tip...

...Workflow Timeline Day 0: Seed and transduce Cells Day 2–3 (am): Remove media, replace with fresh media containing...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...cell death, the cell media should be changed every 2–3 days to maintain the dose of antibiotic, which may...confluent 10 cm dish can be expanded into two 75 cm 2 flasks, etc. *Pro-Tip* This selection method results...with Lentivirus You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service...dilutions and pick the population that has the most desirable level of expression. Over time, transgene expression...

...cooling to room temperature (~45 minutes). Method 2. Place mixed oligos in a PCR tube. Place tube in a... a thermocycler programmed to start at 95°C for 2 minutes. Then, gradually cool to 25°C over 45 minutes...vector with 0.75-6 ng of annealed oligos). Transform 2-3μL into your favorite competent bacteria and plate...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...

... recover, set up overnight growth (Estimated time 2-3 hours) Transformation should be performed at the... day to ensure that growth times are limited. Day 2: Harvest cells and purify DNA (Estimated time 3-4 ... Tubes should contain a total of 5 mL (3 mL SOC + 2 mL transformed Endura from two separate transformations...has been absorbed by the agar. This usually takes 1-2 minutes. *Critical* Be careful not to rip or shred...incubation if needed. Place 100 mL sterile LB at 4 ℃. Day 2 (morning) Before beginning, prechill at least four...pellet. The total weight of each pellet should be ~1-2 g. *Pro-Tip* Make sure to weigh the empty tube beforehand...library during amplification. This protocol is designed to be as general as possible but note that individual...

...each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 ...cells you are using). Put the tubes back on ice for 2 min. Add 250-1,000 μl LB or SOC media (without antibiotic...instead of on ice Reduce step 4 from 20 - 30 mins to 2 mins on ice before heat-shock Shorten or skip the ...Because of this, nearly all plasmids (even those designed for mammalian cell expression) carry both a bacterial...

... plate. Incubate plates at 37 ℃ for 18 hours. Day 2 Observe plates for colony formation. Shown below ...individual colonies and effective selection. 150 µL of 2 mg/mL Carbenicillin plated over-agar Plate shows less...individual colonies with smaller size than the 1 mg/mL and 2 mg/mL plates and effective selection. Selection Curve... Bacterial Culture Introduction This protocol describes methodology for plating antibiotic over-agar for...

...chloride 12.0 g agar-agar 1 L Sterile H 2 O Sterile plates of your desired size - we usually use 60 mm x 15 ...indicated, the antibiotic powder can be dissolved in dH 2 O. *Note: Carbenicillin can be used in place of ampicillin...low a concentration for selection. Negative Result 2: Neither Strains Grows If neither strain grows, it's...next to the flame and begin pouring. Measure your desired amount of agar with a pipete for the first plate...

...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 to ...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...

... overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube or cryovial and gently mix. Note...plasmid DNA, the plasmid will already be in your desired bacterial strain and you will not need to obtain...

...10μL reaction for 5X buffer) 0.5-1μL T4 DNA Ligase H 2 O to a total of 10μL Note: If the DNA concentrations...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 ...

...DNA concentration alone. One method is to conduct 2 ligations for each plasmid you are trying to create...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...restriction enzymes that cut within your insert. Adding desired restriction sites to your recipient plasmid : You...

...DNA concentration alone. One method is to conduct 2 ligations for each plasmid you are trying to create...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...ends of the ORF and how these are used for primer design: Because we are cloning an ORF, we want to clone...TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design is similar, but we need to use the reverse complement...