...of interest (see below for recommend amounts) Appropriate restriction enzyme (see manufacturer's instructions...instructions for proper ammount) Approrpriate restriction digest buffer (see manufacturer's instructions) ...as Addgene's Sequence Analyzer . Determine an appropriate reaction buffer by reading the instructions for...The amount of DNA that you cut depends on your application. A diagnostic digest typically involves ∼500 ...volume usually varies from 10-50 µL depending on application and is largely determined by the volume of DNA...better. Mix gently by pipetting. Incubate tube at appropriate temperature (usually 37 °C) for 1 hour. Always...manufacturer’s instructions. *Pro-Tip* Depending on the application and the amount of DNA in the reaction, incubation...

...PBS, remove the wash, and dispose of it in an appropriate waste container. Fix each well with 500 µL of...as methanol or acetone may be better for some applications. Remove the paraformaldehyde and follow your...safety guidelines for disposing of waste in the appropriate container. Wash 3x for 5 min in 500 µL PBS on...permeabilization buffer and dispose of it in an appropriate waste container. Wash 3x for 5 min in 500 µL ... the blocking buffer and dispose of it in an appropriate waste container. Dilute the primary antibody ...the primary antibody and dispose of it in an appropriate waste container. Wash 3x for 5 min in 500 µL ... at RT in the dark. *Pro-Tip* The plate can be wrapped in foil to block light. Remove the secondary antibody...

...concentration disolved in the appropriate liquid solvent. See table to the right for appropriate antibiotic concentrations...Strains Grow Assuming the appropriate strains were streaked on the appropriate plates, then if both strains... LB-agar powder you’ve measured out into an appropriately sized bottle for autoclaving. We make 400 mL...autoclave. Be sure to check the literature for an appropriate sterilization technique if you are working with... wipe down with a paper towel. Count out the appropriate number of plates and stack them on your lab bench...of molten gel mix, a tube rack containing the appropriate antibiotics, and a section for active pouring...antibiotic. Incubate both plates overnight at the appropriate growth temperature and check for growth. See ...

... thaw on ice (approximately 20-30 mins). Remove agar plates (containing the appropriate antibiotic ) from...spreading device Reagents LB agar plate (with appropriate antibiotic) LB or SOC media Competent cells DNA...Pro-Tip* Transformation efficiencies will be approximately 10-fold lower for ligation of inserts to vectors...transformation onto a 10 cm LB agar plate containing the appropriate antibiotic. *Pro-Tip* We recommend that you plate... take up the DNA, an electro-magnetic field is applied to the cell/DNA mixture to induce membrane permeability... to have access to an electroporator and the appropriate cuvettes. Follow the manufacturer's instructions...

...your convenience. See “warranty information” in appendix. Table of Contents A. pLKO.1-TRC Cloning Vector...References H.1 Published articles H.2 Web resources I. Appendix I.1 Sequence of pLKO.1 TRC-Cloning Vector I.2 ...GenHunter: #Q401 FuGENE® 6 Transfection Reagent Roche Applied Biosciences: #11814443001 OPTI-MEM® serum-free ...protamine sulfate, and puromycin are located in the “Appendix”. F.2. Determining the Optimal Puromycin Concentration...overnight. Day 2: b. The target cells should be approximately 80-90% confluent. c. Dilute puromycin in the...increments. d. Label plates from 1-10 and add appropriate puromycin-containing media to cells. Days 3+:...overnight. Day 2: b. Target cells should be approximately 70% confluent. Change to fresh culture media...

...pool has been sufficiently selected with the appropriate antibiotic so that every cell in the culture ...stable cells such that the next day they will be approximately 50–60% confluent. For 293T cells this is about...confluent or the medium is acidified (i.e., the medium appears orange/yellow if using a standard phenol red pH...counting the cells. When you ultimately transfer approximately 50–100 cells to the conditioned medium, it will...solution at a concentration of 5 cells/mL. Prepare approximately 10 mL of this cell solution for each 96-well...which you see growth. At this time, the cells will appear as colonies in the well ( Figure 1 ). You will ...

...contains the expected size insert. By selecting the appropriate enzyme(s), one can either linearize a plasmid...interest (see below for recommended amounts) Appropriate restriction enzyme (see manufacturer's instructions...instructions for proper amount) Approrpriate restriction digest buffer (see manufacturer's instructions) Gel...others. When choosing restriction enzymes for this approach, it is often a good idea to choose two different...products on a gel and those clones with the appropriately sized products should have the correct orientation...

...container Personal protective equipment (PPE) appropriate for the biosafety level of the lab space Reagents... like the one in the picture above. Wear PPE appropriate for the lab space in which you are working. Even...that the centrifuge is clean and that everything appears to be working smoothly. Place your sample tubes... the centrifuge so that the pellets form in approximately the same place in each sample. Place the centrifuge...according to speed and time (and temperature, if applicable) listed in your protocol. *Pro-Tip* Spin speed...

...than longer ones. Thus, you can determine the approximate length of a DNA fragment by running it on an ... bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock... bands will be differentially intense. If this happens, you can just soak the gel in EtBr solution and...Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. A typical run...are usually referred to as ‘bands’ due to their appearance on the gel. *Pro-Tip* If you will be purifying...

...microcentrifuge. Remove the gel from the plastic wrapper and rinse with deionized water. Gently remove the...5 µL of the prestained protein ladder to the appropriate well. *Pro-Tip* When possible, skip one lane ...µL of each recombinant antibody sample to the appropriate well. *Pro-Tip* Leaving clear lanes between samples... Take a brightfield image of the gel with an appropriate imaging system. Recombinant antibody preps should...Gels . Select Plot Lanes . One graph per lane will appear with peaks representing each protein band. Use ...

...the basics of how to weigh reagents. Wear the appropriate personal protective equipment (PPE) prior to ...contain the reagent while on the balance, and an appropriately sized container to put the reagent in. For some...container. Dispose of the excess material into an appropriate waste container if the material is biohazardous...biohazard solid waste container. Conclusion Appropriately weighing reagents is important to ensure the...

...working in the lab. Do not eat, drink, chew gum, or apply makeup in the laboratory. Before you start your ...and uncluttered. Disinfect your workspace with appropriate materials. For example, you may use 70% ethanol...your lab supervisor what is available and the appropriate contact times for each agent. Make sure you have...autoclaved. Conclusion Although simple, following appropriate BSL-1 and BSL-2 protocol goes a long way. It ...

...expression observed using transient transfection approaches, generating cell lines using lentiviral vectors... tubes, VWR 21008-216 Lentivirus preparation Appropriate antibiotic for selection (e.g. puromycin, blasticidin...lower dilutions depending on your downstream applications. If you’ve titered your virus beforehand, you.... Add 1.5 mL of DMEM complete containing the appropriate antibiotic. This is the beginning of the selection...

...bottom. Add 5 mL of Pierce IgG Elution Buffer to the capped Gravitrap column and incubate for 10 min at RT...subsequent centrifugation steps. *Pro-Tip* Resin will appear compacted after centrifugation. Discard the flow...column in a new 50 mL LoBind collection tube. Slowly apply the sample to the center of the compact resin bed...exchange and concentration with an Amicon column Apply 15 mL of PBS to the reservoir of a Amicon Ultra-...

...sufficient quantities of library for experimental applications. Repeated amplifications should be avoided as... of a library of 100,000 plasmids. Colonies may appear small and require extra incubation time in order... as the quantification method. If all Maxipreps appear to contain sufficient DNA for use, pool samples...purifications. Consult the manufacturers handbooks for appropriate volumes and numbers of tandem purifications. ...

...(with appropriate antibiotic) Bacterial stab Procedure Obtain an LB agar plate with appropriate antibiotic...working near a flame or bunsen burner. Obtain the approrpriate bacterial stab or glycerol stock . Using a sterile...

...add liquid LB to a tube or flask and add the appropriate antibiotic to the correct concentration ( see...Large plasmids usually have a low copy number (approximately one or two copies per cell) and they need to...to grow for longer periods of time (approximately 18-30 hr). On the other hand, smaller plasmids can be...

...protocols that you can use for a wide range of applications, with videos for select protocols in the right-hand...preparations. Antibodies Protocols for common antibody applications. Intro to the Lab Bench Name Description Link... of DNA to a plasmid Gibson Assembly Combine overlapping DNA fragments in a single reaction Ligation Independent...

...see your image through both sides. Once you are happy with the lighting, use the coarse focus knob to ...make minor adjustments to the focus. After you are happy with the positioning and focus of your image on ...microscope after use and store the microscope appropriately....

...overnight culture of bacteria . *Pro-Tip* Refer to appropriate DNA prep protocol for volume of bacteria to grow...the plasmid DNA. Either spin to pellet the DNA or apply the solution to a column that will bind the now ...Aqueous DNA phase Middle phase - A white layer may appear, consisting of precipitated protein particles Bottom...