...reagent you will be weighing out, a weighing boat or weighing paper to contain the reagent while on the...that you are weighing by looking for a weight range on the scale. Make sure that the weight of the material...out the material you are weighing onto the weighing boat or paper. If you weighed out too much material,... up. Discard the weighing boat or weighing paper in the trash if the material weighed is not biohazardous... Protocols Weighing Reagents Weighing Reagents Protocol Intro ... to handling the materials. Equipment Weighing boats or weighing paper Balance Scoopula or spatula Container...button. Before weighing out your reagents, determine the amount that you need to weigh. Gather the reagent...

...The route the light follows from the source to your eyes is called the light path . Light travels from ... Protocols Using a Light Microscope Using a Light Microscope Intro ... to encounter: the compound light microscope. As the name suggests, light microscopes take advantage of...compound light microscope with the main components labeled: Figure 1: Diagram of a compound light microscope...from the light source through a condenser that helps concentrate the light onto the sample, and then through...objective! Once on the higher objective, you will likely need to re-adjust lighting and focus. Increase ...basics of how to use a light microscope. Protocols...

...LB and weigh pellet. The total weight of each pellet should be ~1-2 g. Pro-Tip Make sure to weigh the empty... plates at 30 ℃ overnight. Plate 2.5 mL of the transformed cells on each of the eight bioassay plates ...are often used for screening, barcoding, or other high throughput multiplexed experiments. These pooled... we recommend the use of a diagnostic digest and high-throughput next generation sequencing (NGS). Select...Workflow Timeline Day 1: Transform, recover, set up overnight growth (Estimated time 2-3 hours) Transformation... Alternatives include Stbl4 cells or other ultra-high efficiency electrocompetent cells that are suitable...of electrocompetent cells is essential to ensure high efficiency uptake of plasmid library DNA. This quantity...

...numbers, 50 or more per cell and have a high copy number. High copy number plasmids should only need to...determine if your plasmid is high or low copy. I didn't get any growth after overnight incubation. What went ...However, a liquid culture is capable of supporting a higher density of bacteria and is used to grow up sufficient... The following protocol is for inoculating an overnight culture of liquid LB with bacteria. Video Watch...Prepare liquid LB. For example, to make 400 mL of LB, weigh out the following into a 500 mL glass bottle: 4 ...2 mL in a falcon tube, but for larger preps you might want to use as much as a liter of LB in a 2 L Erlenmeyer... sterile aluminum foil or a cap that is not air tight. Incubate bacterial culture at 37°C for 12-18 hr...

...Introduction Many molecular biology techniques require highly purified and concentrated plasmid DNA. This page...microcentrifuge Desktop vortexer Vacuum (optional) Reagents Overnight culture of bacteria transformed with your plasmid...Protocol: Generalized DNA Purification Grow an overnight culture of bacteria . Pro-Tip Refer to appropriate... with DNA preparation. Pro-Tip If your entire overnight culture cannot fit into a single centrifuge tube...of dH 2 O Store Solution III at 4°C Grow 2 mL overnight cultures from single colonies of bacteria containing...often thought that an incubation of 20 min to overnight at -20 °C or -80 °C will improve precipitation... the tube for 5 min at room temperature on the highest setting. Note: You should see clearly separated...

...least 30 min. The high pressure will prevent your gel mix from boiling over at high temperature. Pro-Tip...your agar is at the right temperature, we recommend using a laser thermometer. Light the flame at the plate...them out at room temperature overnight to allow them to dry. After overnight drying, we place the plates...the appropriate liquid solvent. See table to the right for appropriate antibiotic concentrations. Antibiotic...its cap or aluminum foil (but do not make an air-tight seal!) and tape the bottle with autoclave tape. ... from the autoclave and will cool your gel-mix slightly. Even so, you should always use thermally insulated...resistant to the antibiotic. Incubate both plates overnight at the appropriate growth temperature and check...

...protein, Apolipoprotein L3 (red) overnight, blocked, and incubated overnight with Anti-Desmin [D7]. Plates...protocol may need to be adjusted to accommodate slight differences between products. Addgene does not ...plate seal and incubate at 37 °C for 30 min , or overnight at 4 °C . Section 2: Block the plate Prepare the...set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section 3: Primary antibody incubation...set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section 4: Secondary antibody incubation...set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section 5: TMB reaction Carefully remove...to blue and generally takes between 15 to 30 min. High concentrations of HRP yield a greenish solution....

...Protocol Version 1.0. December 2006. Copyright Addgene 2006, All Rights Reserved. This protocol is provided...ligation, use only long-wavelength UV light. Short wavelength UV light will increase the chance of damaging...culture plate. Incubate cells at 37°C, 5% CO 2 overnight. Although cells should regularly be passaged in... in ten 6 cm plates and grow at 37°C, 5% CO 2 overnight. Day 2: b. The target cells should be approximately...Plate target cells and incubate at 37°C, 5% CO 2 overnight. Day 2: b. Target cells should be approximately...plate, add between 0.05-1 mL virus (add 0.5 mL for a high MOI, and 0.1 mL for a low MOI). Scale the amount...cell line. d. Incubate cells at 37°C, 5% CO 2 overnight. Day 3: e. Change to fresh media 24 hours after...

...PEI μg ratios provided high transfection efficiencies as measured by the highest proportion of GFP positive...without limiting cell growth. Left panels: bright field images; right panels: GFP channel images. Last reviewed...meter Stir plate Magnetic Stir Bar Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine ...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and ...the packaging cell line is critical for obtaining high viral titer. 293T cells should be split 3 times ...purification should include an endotoxin removal step. For high quality plasmid DNA, the plasmids should also be...transfection to determine what ratio gives the highest percentage of GFP positive cells. Refer to the ...

...Casting tray Well combs Voltage source Gel box UV light source Microwave Reagents TAE ( recipe here ) Agarose...you to visualize the DNA under ultraviolet (UV) light. Caution EtBr is a known mutagen. Wear a lab coat...gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the density... in the first place. Carefully load a molecular weight ladder into the first lane of the gel. Note: When...the second stop and carefully raise the pipette straight out of the buffer. Carefully load your samples...destain for 5 mins. Using any device that has UV light, visualize your DNA fragments. The fragments of ...minimize damage to the DNA. Note: When using UV light, protect your skin by wearing safety goggles or ...

...are having trouble getting colonies, you might want to use higher competency cells. Additionally, if your...amplify your insert DNA. It is important to use a high fidelity taq polymerase to minimize mutations. The...the digest goes at least 4 hours and as long as overnight. If you are going to use only one restriction ...final product is going to be very large (>10kb) you might want to use electro-competent cells instead of the...insert plate that are not correct. If you have a high number of colonies on your recipient plasmid alone...more colonies you will need to pick) and grow overnight cultures for DNA purification. After purifying... by Sequencing PCR based cloning carries a much higher risk for mutation than restriction enzyme based...

...controller Hazardous waste container Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine ...alternative such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5...health of the cell line is critical for obtaining high levels of virus. HEK293T cells should be split 3...purification should include an endotoxin removal step. For high quality plasmid DNA, the plasmid should also be ...transfection to determine what ratio gives the highest percentage of GFP positive cells. Refer to the ...pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided high transfection efficiencies as can be seen here by...green fluorescent protein expression (green in the right panels) with a limited effect on cell growth. Last...

...are having trouble getting colonies, you might want to use higher competency cells. Additionally, if your... on your gene of interest (YGOI for short). You might need to express YGOI in cultured mammalian cells...the digest go at least 4 hours and as long as overnight. If you are going to use only one restriction ...final product is going to be very large (>10kb) you might want to use electro-competent cells instead of the...insert plate that are not correct. If you have a high number of colonies on your recipient plasmid alone...more colonies you will need to pick) and grow overnight cultures for DNA purification. After purifying...

...significantly higher density than water, then you may need to fill the blank based on weight instead of ..., which is generated by spinning the sample at a high speed. Being able to separate solids from a liquid...Centrifuges sit on the floor and can be about bench height. They often spin at similar speeds to their smaller...larger containers, but they can also rotate at much higher speeds and are used for more specialized techniques...distributed evenly around the center (see image to right). Even distribution ensures that the centrifuge ...

...will often get higher transformation efficiencies with less DNA, especially when using highly competent cells...transformation efficiencies upon thawing. For the highest transformation efficiency, we recommend that you... for the purposes of storage and amplification. Higher efficiency cells are more important if you will...won't grow in colonies. Incubate plates at 37°C overnight. Tips and FAQ How can I save time when carrying...reduce the efficiency of the transformation, so when higher efficiency is needed follow the complete protocol...

...Use a "Universal" SYBR master mix which contains a high-quality DNA polymerase and a blend of dTTP/dUTP ... use plasmid #59462 from Addgene. The values highlighted below in red were calculated using this plasmid...of Addgene plasmid #59462 : 1.07 μg/μL Molecular Weight: 6208 bp x 650 dalton/bp or (g/mole)/bp = 4.03 ...Dilution 8 20 uL Dil. 7 80 uL 5X 3125000X Dilutions highlighted in green are the ones loaded onto the qPCR plate...

...acrylamide when immunoblotting high molecular weight proteins and higher percentage acrylamide when immunoblotting...protocol may need to be adjusted to accommodate slight differences between products. Addgene does not ...immunoblotting low molecular weight proteins. This protocol uses a dry transfer device but can be adapted...typically between 1–10 μg/mL. Incubate the membrane overnight in primary antibody at 4 °C on a rocking platform...

...subtract the weight of the empty tube from the weight of the tube with the gel fragment. The weight of the ...gel in a labeled microfuge tube. Using a scale, weigh the tube with the gel fragment after zeroing the... the gel percentage to get better separation. A higher percentage agarose gel will help resolve smaller...

...sample Using a Light Microscope Learn about the parts of a light microscope and its use Weighing Reagents Learn...applications, with videos for select protocols in the right-hand column. You can find even more video content... Learn how to weigh laboratory materials on a balance Basic Molecular Biology Name Description (Link opens...

...controller Hazardous waste container Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine ...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5mL...sample of possible dilutions. You may want to try higher/lower dilutions depending on your downstream applications...transduced with lower dilutions of the virus will have higher levels of expression. Consider expanding populations...population may drop. This is because cells that express high levels of the transgene may have reduced growth ...

...counter Reagents Polyclonal stable cell pool DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine ...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5...for one 96-well plate. Allow the cells to grow overnight. Replace the medium on the cells and collect the...conditioned medium, it will be more accurate if a higher volume of homogenized cell solution is transferred... Western blotting to screen for lines with the highest or lowest transgene expression ( Figure 2 ). Sample...

...PES. Using a PES filter will maximize titer . DMEM high glucose, Corning 10-013-CV DMEM, low glucose (1 ...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5...dissolved. Pro-Tip This step is challenging due to the high viscosity of PEG. Stirring under medium heat will...precipitation. Precipitation of the viruses can proceed overnight at 4 °C if needed. Transfer the entire sample ... prep . The clarified supernatant can be kept overnight at 4 °C before proceeding with the purification...

...Inoculating an Overnight Liquid Culture . After you have bacterial growth, add 500 μL of the overnight culture...bacteria onto an LB agar plate . Grow your bacteria overnight at the appropriate temperature. Growth conditions...page. The next day you will be able to start an overnight culture for plasmid DNA prep the following day...

...container Inverted fluorescence microscope Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine ...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5...each well of the 6-well dish. Incubate the cells overnight. If using freshly collected virus, filter through...If you anticipate that your viral stock will be higher titer, consider additional dilutions. Dilution ...

...protocol may need to be adjusted to accommodate slight differences between products. Addgene does not ...mg/mL DAPI solution to 100 µL PBS. Protect from light. Prepare a 300 nM DAPI working solution by diluting...DAPI stock solution into 5 mL PBS. Protect from light. Procedure Section 1: Seeding cells Place a sterile...Pro-Tip The plate can be wrapped in foil to block light. Remove the secondary antibody and dispose of it...

...controller Hazardous waste container Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine ...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat inactivated FBS and 5...colonies for at least 2 of the dilutions. Pro-Tip The higher dilution wells tend to provide more accurate titers...dilutions, and single colonies are visible in the higher dilutions. Last reviewed on: August 31, 2023...

...to begin using the pipette. To the right is a photo of how you might hold a pipette. Without moving past... P1000 pipette shows the volume adjustment ring right below the plunger. This ring changes the pipette...P20, P100, P200, and P1000 as shown from left to right. Pipette Dispense Volume P2 0.2 to 2 µL P10 1 to...

...purified AAV. Option #3 Puncture the QuickSeal tube slightly below the 60–40% interface with an 18 ga needle... (1999): 973-985 (Link opens in a new window) . Right panel: cartoon indicating the position of the needle...harvesting of the purified AAV using option #1. Right image adapted by permission from Macmillan Publishers... viral vectors," (Link opens in a new window) copyright (2006). Figure 3: Example of an SDS-PAGE gel of...

...viral particles. Users may need to run lower or higher dilutions depending on their particular sample....tissue culture treated dish, VWR, 29442-042 DMEM, high glucose, pyruvate, Corning, 10-013-CV Heat-inactivated... Reagent Preparation DMEM Complete: 500 mL DMEM, high glucose, pyruvate 55 mL heat inactivated premium...increases by a factor of ~2 as you progress from the higher dilutions to the lower dilutions (blue). As shown...

...spectrophotometer uses the absorbance/transmission of light through a liquid to determine the concentration ...liquid. Molecules absorb different wavelengths of light to varying degrees and most have a specific wavelength... absorbance, you need to know the wavelength of light that your substance maximally absorbs. In the case...