...move around pieces of DNA. Oftentimes several approaches will work for any specific cloning project; however...likely that for any given project there is an ideal approach. This may be due to speed, cost, availability ... of time required for cloning. To utilize this approach, the fragment of DNA that you would like to clone...amplified with specific Gateway attB1 and attB2 sites attached to the 5’ and 3’ ends of DNA sequence. This fragment...fragments generating long overhangs that anneal to each other due to their homology. DNA polymerase then... enzyme cloning. Scientists have utilized this approach to create compatible custom overhangs that can...

...cDNA libraries, each plasmid contains a unique cDNA. In shRNA or gRNA libraries, each plasmid contains...sequence, but there are multiple sequences targeting each gene in the overall library. Barcoding libraries...negative results - for a given plasmid, think of each cell carrying it as being a biological replicate...

...engineered α7 nicotinic acetylcholine receptor (nAChR) domains that respond to specific small molecules...Adeno-associated virus (AAV) is a widely-used tool for achieving in vivo expression of chemogenetic receptors, ... our AAV guide . FLEx Vectors are also used to achieve cell-specific expression of AAV-encoded chemogenetic...Azua IR, Li JH, et al (2009). A chemical-genetic approach to study G protein regulation of β cell function...

...produce AAV in the lab. Addgene’s AAV Guide will teach you about AAV components, serotypes, pseudotyping...molecular weight structures that are maintained extrachromosomally. rAAV are more likely than wild-type AAV ...the virus depends on the cell’s DNA replication machinery to synthesize the complementary strand, transgene...

...creating replication-competent virus increases with each round of amplification. What is RCA? RCA stands ... probability of a crossover event increases with each round of amplification. Where can I find information...

...plasmids can be used in transient transfections to achieve expression of the transgene, and those that can...the insert from a parent vector, some possible approaches to using this site include subcloning or appending...

...guide to viral vectors contains an introduction to each vector and its applications, tips for using vectors...

...RNA polymerase in binding to the promoter region. Each sigma factor recognizes different core promoter ...

...-3 minutes for cells to detach. Gently tap the sides of the CS2 to help detach the cells, add 200 mL of...determine the total μg/bp we need to achieve a 1:1:1 molar ratio of each plasmid: 2000 μg / 24,961 bp = 0.08... minutes on ice between each pulse, 50% amplitude. Return to ice between each round of sonication to avoid...* While adherent, these cells are very loosely attached to the dish surface and should be handled carefully...magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) 1 mg/mL Polyethylenimine (...the maintenance phase and do not allow cells to reach 100% confluence (80–90% is ideal). Pass and plate... flasks. Cells should be at ~80% confluence. For each T-175 flask: Aspirate culture media and rinse once...

...master mix to add to each PCR tube. Secure the tops to the PCR tubes, gently tap each tube to bring all ...DMSO to each reaction. The ideal setup for this troubleshooting step is to do one reaction with each, and...stable within this temperature range to anneal to each of the single stranded DNA template strands. The...synthesized strands. Materials List Reagents for each 50µL PCR : Thin-walled PCR tube Ice Bucket 2 μL ... 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 mM each nt) 2.5 μL Forward Primer (10 μM stock) 2.5 μL Reverse...2 O 0.2 μL Taq DNA Polymerase (5 units/μL) PCR Machine Agarose Gel Procedure Primer Design and PCR Design...

...aspirate, using a new aspirating pipette for each well. Detach cells by incubating with 200 µL TrypLE for 1...concentration of each sample on a spectrophotometer. Prepare 25 ng/µL stocks of each sample. Samples can...use. Wipe down all pipettes and surfaces with 10% bleach. Safety Warnings Lentivirus is generally considered...magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) Trypsin EDTA (TrypLE, Thermo... of 80-fold dilution 200 µL 400 µL Add 150 µL of each viral dilution to a well of a 6-well plate (1 dilution...the final dilutions range from 25–1600-fold. Mix each well with a 1 mL pipette 5–10 times. The final volume...plates. Be sure to use a new aspirating pipette for each well as to not contaminate cell conditions with ...

...conical tubes as needed. Add 10 mL cold LB to each plate for each scrape and use spreader to scrape plates..... Add each scrape into a 50 mL conical tube on ice pooling the scrapings of two plates into each tube....tube. Keep each tube on ice while scraping. Repeat addition of 10 mL cold LB and scrape for each plate until...adequate quality, but this upfront cost will save headaches and expense later. At a minimum we recommend the... tips in a -20 °C freezer. Aliquot 3 mL SOC into each of four 14 mL Vented Falcon Tubes and have 1 mL ...for all steps. Procedure Day 1 Add 200 ng DNA to each 50 µL aliquot of thawed Endura Duos on ice. Flick...vented Falcon Tube containing 3 mL SOC. Repeat for each of the 25 µl aliquots of cell/DNA mixture used (...

... µL of 4X sample buffer to each sample. Add 2 µL 10X reducing agent to each sample. Spin the sample briefly...peaks representing each protein band. Use the line tool to connect the bottom of each peak. Select the ...µL of 0.25 mg/mL with 10 µL PBS Transfer 5 µL of each standard to a microfuge tube. Add 8 µL of PBS to...gel. Carefully remove the comb from the gel. Rinse each well with 200 µL 1X MOPS running buffer. Fill the...skip one lane before loading samples. Load 20 µL of each recombinant antibody sample to the appropriate well...gel easier. Cover the electrophoresis chamber and attach to a power supply. Run the gel at 150 V for 1 h...Select Image . Select Type . Choose 8-bit . Determine each lane of the gel as follows: Figure 2 Using the box...

...the cells have detached from the plate and can be isolated. When trypsinized, detached cells will exhibit...the transgene, and the specific conditions used. Each 96-well plate described in this protocol could, ...on the A549 cells described in our sample data , each 96-well plate gave rise to 15–20 monoclonal lines...cells this is about 2 × 10 6 cells in a 10 cm dish. Each 10 cm dish should be seeded in 10 mL DMEM complete...Prepare approximately 10 mL of this cell solution for each 96-well plate you plan to seed. If you chose not... Transfer 100 µL of the 5 cells/mL solution into each well of a 96-well plate. By doing this, you are ...any well receives more than one cell. *Pro-Tip* In each 96-well plate, seed one of the corner wells with...

...497 Add 0.5 mL of a single viral dilution to each well (each well gets one dilution, so a 6-well plate will...50,000 cells) into each well of the 6-well dish. This brings the total volume in each well up to 1.5 mL...expression observed using transient transfection approaches, generating cell lines using lentiviral vectors... calcium or magnesium (cations can affect the attachment of adherent cells) Microcentrifuge tubes 6-well...reverse transduction" by seeding 50,000 cells into each well of the 6-well dish. These cells will be added...polybrene, the final concentration of polybrene in each well should be 10 µg/mL. *Pro-Tip* Transducing too...which may not be stable at 37 °C. *Pro-Tip* To achieve a stable cell pool, the antibiotic selection should...

... 96-well plate: Load 5 μL of each standard in duplicate Load 5 μL of each sample in duplicate. Do not ...well by pipetting back and forth multiple times at each step Reagent Preparation Master Mix: count the number...that there is enough master mix for all samples). Each sample requires 15 μL of master mix. *Pro-Tips* ...standard curve is obtained, make a small aliquot of each standard (enough for 1 or 2 plates) and store at...use within 1 week. Keep track of the Ct value for each standard over time. They should remain within 0.5...carrier DNA to a final concentration of 4 ug/mL to each standard dilution. Dilute DNase-treated and AAV ... dilution series is critical. Make sure to pipet each dilution up and down at least 10 times, and use ...

...minutes at 95°C in a PCR machine or in a beaker of boiling water. If using a PCR machine, incubate the sample...recommends that you select multiple target sequences for each gene. Some sequences will be more effective than... for Inserts Day 1: 1. Inoculate 5 colonies from each ligation into LB + 100 μg/mL ampicillin or carbenicillin...different amounts of virus as needed. Day 1: a. For each plasmid to be transfected, plate 7×105 HEK-293T ... NOT use polystyrene tubes), make a cocktail for each transfection: 1 μg pLKO.1 shRNA plasmid 750 ng psPAX2...amount of Fugene® and OPTI-MEM necessary given that each reaction will require 6 μL FuGENE® + 74 μL OPTI-...temperature. e. Add 80 μL of FuGENE® master mix to each tube from step c for a total volume of 100 μL. Pipette...

...use. Wipe down all pipettes and surfaces with 10% bleach. Safety Warnings AAV is generally considered biosafety...place on ice. Wipe down a DG8 cartridge holder with bleach and place in the Biological Safety Cabinet (BSC... a single channel 1–10 µL pipette to add 5 µL of each viral sample to Dilution 1 in the 48-well dilution...avoid generating aerosols. Be sure to change tips each time. Dilution 1 (20X): 5 µL in 95 µL 1X PCR buffer...well freezer block. Add 20 µL of the master mix to each PCR tube. Be careful to dispense to the bottom of...to get to temperature. Once the temperature is reached, place the PCR plate with the foil onto the metal...the following equation to calculate the titer for each sample: $$T = {R*C*D*1000\over V}$$ Where: T = Titer...

...Starting Wipe down all pipettes and equipment with 10% bleach prior to use. Warm the affinity column, binding...gently pour off the ethanol storage buffer. Tightly attach LabMate PD10 Buffer Reservoirs to the top of the...columns, refer to the manufacturer’s instructions. Attach the Gravitrap column to a clamp on a clamp stand...s to mix. Determine the protein concentration of each fraction on the NanoDrop Spectrophotometer using... 3x , discarding buffer from the collection tube each time. Place column in a new 50 mL LoBind collection...NanoDrop Spectrophotometer to see if the sample has reached the desired concentration. If the sample concentration...until the volume of the sample is reduced enough to reach the desired concentration. *Pro-Tip* Periodically...

... Although each pipette performs the same function, the numbers are read differently on each type of pipette...to 100 µL P200 20 to 200 µL P1000 100 to 1,000 µL Each pipette should be used with the appropriate tips...This section will help you understand how to read each display to dispense the correct amount of liquid... picture above), while 0.5 µL would read as 050. Each volume display on these pipettes will also have ...to expel excess liquid from the tip once you’ve reached step 8. Continuing to hold down the plunger, gently...liquid so that it's easier for the pipette tip to reach the liquid. Aim to let only the pipette tip into...