...effective method for the assembly of plasmids, and molecular biologists now use this method extensively...hundred kilobases. Nature Methods , 6(5), 343–345. https://doi.org/10.1038/nmeth.1318 (Link opens in a new...mitochondrial genome. Nature Methods , 7(11), 901–903. https://doi.org/10.1038/nmeth.1515 (Link opens in a new... Assembly Cloning You may also like... Cloning Methods Empty Backbone Guide Molecular Biology Protocols...the J. Craig Venter Institute developed a novel method for the easy assembly of multiple linear DNA fragments...intervening sequence between two PCR products, one method is to “stitch” together several oligos. This technique...

...purity. This can be done with a spectrophotometer. A spectrophotometer uses the absorbance/transmission... instructions for the spectrophotometer specific to your lab. Spectrophotometer Tips Before measuring ...labs have a NanoDrop, which is a very small spectrophotometer that can accurately read DNA concentration...measuring any samples, be sure to ‘blank’ the spectrophotometer using the solution the DNA is resuspended in, ...NanoDrop. It is a good idea to re-zero any spectrophotometer between samples if they are expected to vary...

... we list the specific equipment, reagents, and methods that we use in our lab at Addgene. Equipment and...compatible with 50 mL conical tubes NanoDrop spectrophotometer 37 °C, 5% CO 2 incubator with shaking platform...concentration of each fraction on the NanoDrop Spectrophotometer using the A280 IgG setting. Combine all eluates...concentration of the pooled sample on the NanoDrop Spectrophotometer using the A280 IgG setting. If the concentration...Determine antibody concentration on the NanoDrop Spectrophotometer. Dilute antibody to 1 mg/mL with PBS if needed...to check the concentration on the NanoDrop Spectrophotometer to see if the sample has reached the desired...Periodically check the concentration on the NanoDrop Spectrophotometer to see if the sample has reached the desired...

...be the best fixation method for all. Alternative fixation methods such as methanol or acetone may be better... target protein using the formaldehyde fixation method. The protocol may need to be optimized for different... we list the specific equipment, reagents, and methods that we use in our lab at Addgene. Equipment and.... Tips and Troubleshooting The optimal fixation method will vary depending on the sample type and the ...interest. You may need to try a variety of fixation methods to find the best conditions for your target. The...

...likely to retain stable transgene expression. Other methods of generating a monoclonal cell line include single-cell...controller Hazardous waste container Microscope Hemocytometer or other cell counter Reagents Polyclonal stable... growth of cells in individual cultures. Other methods for enhancing single cell growth, such as using...concentration in this homogenized cell solution using a hemocytometer or other cell counter. *Pro-Tip* Prepare a relatively...well plate. Sample Calculation First, use a hemocytometer to quantitate the homogenized cell solution....screening. Sanjana NE, Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8):783-4. (Link opens in a new window...

...the dilution factors (method 1) or the volume of virus (method 2): Method 1 Method 2 $$T = {N*F*D\over ...with less than 40% fluorescent cells. Titering methods assume one integration event per cell. When the...

...of the device. Select the desired method and make sure the parameters are correct. Transfer for 5–6 min... we list the specific equipment, reagents, and methods that we use in our lab at Addgene. Equipment and...Pipette controller Pipette tips and pipettes Spectrophotometer Heat block Mini gel tank chamber Power supply...using a Pierce BCA Assay Kit or other preferred method for protein determination. Prepare a 50:1 Reagent...

...Titer by a Novel Droplet Digital PCR Method. Hum Gene Ther Methods . 2018 Apr;29(2):96-103. doi: 10.1089...Determine the concentration of each sample on a spectrophotometer. Prepare 25 ng/µL stocks of each sample. Samples...reached, place the PCR plate with the foil onto the metal support block. Place the block in the plate sealer...thermocycling. Thermal Cycling Run the following PCR parameters: Cycling Step Temperature (°C) Time (min) Ramp...

...that it isn't methylation sensitive . Plasmids grown in Dam or Dcm methylase positive strains...sites. See NEB's table of methylation sensitive restriction sites . Sometimes enzymes cut sequences which...

...is added to clearly distinguish the steps. This method will help enrich the prep with full (genome containing...adeno-associated virus purification using novel methods improves infectious titer and yield." Gene therapy...Wolfgang, and Lamla Thorsten. Human Gene Therapy Methods. August 2015, 26(4): 147-157. doi:10.1089/hgtb.2015.051...

...the first step toward efficient gene silencing. Methods for target selection are continuously being improved...and Transforming into Bacteria Use your ligation method of choice. For a standard T4 ligation, mix: 2 μL...Recommended Materials Material Vendor and catalog # Hexadimethrine bromide (polybrene)* Sigma-Aldrich: #H9268 ... et. al. Science . 1996 272:263-267. PubMed . Asymmetry in the assembly of the RNAi enzyme complex. . ...mL antibiotic. Pour plates and store at 4°C. Hexadimethrine Bromide (Polybrene) Prepare a 1mg/mL solution...

...Culture Introduction This protocol describes methodology for plating antibiotic over-agar for the selection...other small flame source) Incubator Reagents 6 cm diameter LB/agar plates without antibiotic High concentration.... It is, however, more expensive. With a 6 cm diameter petri plate containing solidified LB-agar, pipette...

...Efficient annealing can be achieved by one of two methods: Method 1. Place the mixed oligos in a 1.5mL microfuge...slow cooling to room temperature (~45 minutes). Method 2. Place mixed oligos in a PCR tube. Place tube...

... or 70% ethanol Distilled water Disinfectant Thermometer Water bath weights and floats Reagents None needed... example, number of drops per liter. Place a thermometer in the water bath. Oftentimes, the water bath...bath will have a spot to secure the thermometer in place. Turn the water bath on and set the appropriate...

...your ocular lens. For example, if you observe something using the 10x objective and your ocular lens is...a slide, you can secure it into place using the metal clips on the stage. Turn on the power source and...drawing skills. Regardless of your image capture method, be sure to record the magnification used, any ...

... we list the specific equipment, reagents, and methods that we use in our lab at Addgene. Equipment and...chamber Power supply Rocking platform Fume hood Metal spatula Razor blade Plastic tray Gel imaging system...chamber. Remove the gel from the chamber. Use the metal spatula to gently break the gel cast open. Use a...

... pipette 200–1000 µL single channel pipette Hemocytometer or cell counter Ice bucket CO 2 incubator Pipet...Pipet controller Hazardous waste container pH meter Stir plate Magnetic stir bar Sonicator Ear protection...suspension (no clumps). Count cells using a hemocytometer or cell counter. Seed 350 million cells from...

...concentrations of DNA. In our hands fluorescent dye methods tend to report lower values than Nanodrop, although...been designed with Nanodrop as the quantification method. If all Maxipreps appear to contain sufficient ...Maxipreps as yield can dramatically plateau and sometimes fall off entirely if the column or reagents are...

...restriction digests have become the most widely used method scientists employ to selectively move a specific...Although this is never an ideal cloning strategy, sometimes it cannot be avoided. If you do have to do so,...

...Independent Cloning You may also like... Cloning Methods Empty Backbone Guide Molecular Biology Protocols...time-consuming ligation step of traditional cloning methods. In traditional cloning, base-pairing in the short...