... Protocols Weighing Reagents Weighing Reagents Protocol Intro to the Lab...how to obtain the correct amounts of materials for an experiment or reagents for a stock mixture is an...basics of how to weigh reagents. Protocols...get started in the lab! Introduction For many experiments, you’ll need to make buffers, media, or other...of this task is making sure you’re weighing all reagents precisely to create the solution you expect. Understanding...essential laboratory technique, as imprecise measurements can affect the outcome of your experiment. In...protocol, you will learn the basics of how to weigh reagents. Wear the appropriate personal protective equipment...

... 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...total + 10% (3.5μl) = 38.5μl dNTP mix (10 mM each nt): 1µl x 7 reactions = 7µl total + 10% (0.7µl) = 7.7µl...thousands of identical copies using a simple set of reagents and a basic heating and cooling (denaturing and...of the DNA segment to be amplified. When these reagents are combined in an appropriate buffer, a series... the newly synthesized strands. Materials List Reagents for each 50µL PCR: Thin-walled PCR tube Ice Bucket... on ice Set up a 50 μL reaction (Keep all your reagents on ice): 2 μL Template DNA (10 ng-500 ng) 5 μl...

...Join almost any two fragments regardless of sequence. No scar between joined fragments. Fewer steps. One...about what DNA segments you will need to join to create your final plasmid. Adjacent segments should have...If there are significant amounts of undesired product, gel-purify DNA segments. Otherwise, PCR purification...combine segments in the Gibson Assembly Reaction. Pro-Tip Yields will be best when the DNA fragments are ...annealed DNA fragments. Taq DNA Ligase - covalently joins the annealed complementary DNA fragments, removing... al. , 2010). Number of fragments assembled simultaneously Multiple fragments can be assembled in one ... for the easy assembly of multiple linear DNA fragments (Gibson et al. , 2009). Regardless of fragment...

...bottom. For the P1000, this represents the ones. For the P200, this represents the first decimal place. ...For the P20, this represents the second decimal place, and for the P2 this represents the third decimal...in many scenarios to accurately dispense small amounts of liquid (think: 0.1 µL to 1 mL). When working...microfuge tube, bottle, etc.) Labels for containers Reagents Liquid for pipetting Background Information Anatomy... container in order to avoid contaminating the contents. Once the end of the tip is submerged, slowly ...small changes in the volume could affect your experiments. Beyond knowing the proper way to pipette, it...they are properly tuned to dispense the correct amounts of liquid. If you are pipetting the same amount...

...selection in your experiments. (You may wish to repeat this titration with finer increments of puromycin to...See “warranty information” in appendix. Table of Contents A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium...microcentrifuge tube. TIP: When visualizing DNA fragments to be used for ligation, use only long-wavelength...products on a 1% agarose gel. You should see two fragments, a 2kb fragment and a 5kb fragment. 5. Sequence...the host genome. For stable loss-of-function experiments, Addgene recommends that you generate lentiviral...The protocol can be scaled to produce different amounts of virus as needed. Day 1: a. For each plasmid ...puromycin should be from 1-10 μg/mL in 1 μg/mL increments. d. Label plates from 1-10 and add appropriate...

... that has UV light, visualize your DNA fragments. The fragments of DNA are usually referred to as ‘bands...matrix toward a positive electrode. Shorter DNA fragments migrate through the gel more quickly than longer...gel alongside a DNA ladder (a collection of DNA fragments of known lengths). Last Update: Feb. 20, 2018 ...Voltage source Gel box UV light source Microwave Reagents TAE ( recipe here ) Agarose Ethidum bromide (stock...Another method for visualizing very short DNA fragments is polyacrylamide gel electrophoresis (PAGE), ...which is typically used to separate 5 - 500 bp fragments. How do you get better separation of bands? If...

...specifically uses primers and probes targeting the ITR elements in the viral vectors but can be modified for other...GC/mL, where GC is genome copies and generally represents the physical number of viral particles. Users...Before Starting To reduce the risk of contaminating reagents we recommend making small aliquots of master mixes...10199-452 Ice bucket 96-well freezer blocks (x 3) Reagents Molecular Biology Grade Water, Hyclone, SH30538.02...generation BSC) to ensure no contamination of the PCR reagents and the NTC with virus. Pre-warm the 96-well plate...equipment with 10% bleach prior to use and keeping all reagents and samples on ice or pre-chilled 96-well freezer...accuracy of the titer, take the average of several measurements. Figure 1: ddPCR sample data Sample Dilution...

...analyze the resulting fragments by gel electrophoresis . The pattern of the fragments on the gel can indicate... tips Reagents Liquid DNA aliquot of your plasmid of interest (see below for recommended amounts) Appropriate...plasmid into 3-8 pieces such that all (or most) fragments are small enough to be accurately sized on a gel...enzymes that will cut your plasmid into multiple fragments, you can get a very unique pattern that will distinguish...

...rearrangement of the plasmid DNA. Additionally, specific treatments have been discovered that increase the transformation...important if you will be transforming with very small amounts of DNA or if you're multiple plasmids at once. ...Microcentrifuge tubes Sterile spreading device Reagents LB agar plate (with appropriate antibiotic) LB...colonies, while allowing you to recover all transformants. If the culture volume is too big, gently collect... at least 20-30 mins) I get very few if any transformants when transforming large plasmids (>10 kb) or...manufacturer's instructions for each. I got no transformants. What went wrong? Check that you are plating...

...electron microscopes that allow us to see cellular components with science-fiction levels of detail. You can... of a compound light microscope with the main components labeled: Figure 1: Diagram of a compound light... make it through the sample because different components of the specimen will refract and reflect the ...) using dyes or stains that add color to some components of the sample but not others. The light that ...other sample that can fit on a microscope stage) Reagents None needed Procedure Set up your microscope by...the smaller of the focus knobs) to make minor adjustments to the focus. After you are happy with the positioning...

...invert the tube carefully 5 times to mix the contents. The contents will become clear and thicker as the proteins...microcentrifuge Desktop vortexer Vacuum (optional) Reagents Overnight culture of bacteria transformed with...step causes the bacteria to lyse, releasing their contents, including plasmid DNA, into solution. Add a renaturing...,000 g. Notes: Pellet contains proteins, cell fragments, salt and other extra particles from solutions... This will help to remove proteins and other contaminants from the plasmid DNA. It is also advisable to...

...barcoding, or other high throughput multiplexed experiments. These pooled plasmids differ by only a short...resulting in a smaller plasmid containing only the elements required for bacterial propagation (origin of ...Electroporator (MicroPulser TM , Bio-Rad 1652100) Reagents 200 µL electrocompetent cells (Default: 4 tubes...of cells. Ensure access to autoclaved, sterile reagents for all steps. Procedure Day 1 Add 200 ng DNA ...cells, more DNA, gentler practices, and colder reagents (not including the recovery media!). Do not proceed...and sometimes fall off entirely if the column or reagents are significantly overloaded. If you find that...

... connecting the sugar backbone of the two DNA fragments. This reaction, called ligation, is performed ...The majority of ligation reactions involve DNA fragments that have been generated by restriction enzyme...insert will be added in the correct orientation and prevents the vector from ligating to itself during the ...phosphatase removes the 5' phosphate and therefore prevents the ligase from being able to fuse the two ends...of intact plasmid in ligation or transformation reagents Optimizing the Vector:Insert Ratio Although a ...

...protocol is composed of steps that separate out contaminants from an impure AAV preparation. The 15% iodixanol...macromolecules. The 40% and 25% steps are used to remove contaminants with lower densities, including empty capsids...Equipment Ultracentrifuge T70i rotor or equivalent Reagents Opti-Prep (60% Iodixanol) QuickSeal tubes QuickSeal...have a good idea when the virus elutes from the gradients. In a biosafety cabinet, carefully puncture the...by silver stain. Note the increased number of contaminants in each fraction. * AAV capsid proteins VP1,...

...gloves, and whenever possible, keep RNA sample and reagents cold and work quickly to reduce RNA degradation...degradation. Keep work area, equipment, and reagents RNase-free (Use an RNase decontamination solution, such as...Homogenizer Vortexer -20°C freezer -80°C freezer Reagents Solution D (for Protocol Option #1 ): 4 M Guanidinium...Sheet) for safety warnings and hazards for these reagents. Work in a well-ventilated space and under a fume... fume hood when working with the volatile reagents in the list above. Procedure Option #1 - Solution D...

...potential accidents such as spills. BSL-2 is different because it includes labs that work with agents associated...don’t touch your face! Close-toed shoes and long pants should be worn to protect your legs and feet from...wear glasses/goggles in addition to the BSL-1 requirements. Conclusion Although simple, following appropriate...

... and using a centrifuge to separate different components in a liquid sample....laboratory equipment used to separate different components in a liquid sample. It achieves this by using...appropriate for the biosafety level of the lab space Reagents Sample to be centrifuged Water Background Information... you use, most will have essentially the same components. The photo to the left shows a front view of ...

... and using a centrifuge to separate different components in a liquid sample Using a Light Microscope Learn...parts of a light microscope and its use Weighing Reagents Learn how to weigh laboratory materials on a balance...plasmid Gibson Assembly Combine overlapping DNA fragments in a single reaction Ligation Independent Cloning...Lentivirus Quantify virus using fluorescence measurements Colony Formation Titering Assay for Lentivirus...

...equipment, reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other ...channel pipette 200–1000 µL single channel pipette Reagents 1X PBS Microcentrifuge tubes Sterile Poly-D-lysine..., incubation times, and recommended compatible reagents. Secondary antibodies must match the host species...

...therefore, the cells cannot be used for later experiments. Workflow Timeline Day 0: Seed and transduce ...incubator Pipet controller Hazardous waste container Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-...lines. It is not recommended that lentiviral supernatants be subjected to multiple freeze-thaw cycles....titers as the likelihood of multiple integration events decreases as the dilution increases. Calculate ...