...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...

... homepage! You can track the number of plasmids right below the search bar and more stats further down...

...a: That will warm up the entire lab (hint: you might need some mini marshmallows…). We’ll choose 4 submissions...

...! Our mascot who has supported us over the past eight years now has a new friend to join them. Blugene...

...starting their careers in the lab and even those who might just need a refresher on some best practices for...

... annual #DeckTheLab contest. The bar is set very high from last year’s impressive entries, but we have...

... to me… Antibodies developed by IPI!   🎶On the eighth day of Antibody, Addgene gave to me…  A recombinant...

...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 ...

...Segment on mCyRFP1 Hot Plasmid 4: mScarlet - a new, bright, and monmeric red fluorescent protein.  Listen ...

...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...

...editing by a miniature CRISPR-Cas12f nuclease. On the eighth day of CRISPR, Addgene gave to me: the PEDAR strategy...

...annual #Deckthelab contest. The bar was set very high from last year’s impressive entries, but our community...

...Goedhart  Created in collaboration with Addgene writers High Complexity Golden Gate Assembly with NEB's DADExperts...

...with you.  Top 5 science comics:   1)   “Piled Higher and Deeper” by Jorge Cham   2)   “Boxplot” by Maki...

...in 8,900 institutions across 111 countries. Just eight days into 2024, we've already shipped 2,078 items...

...journey. And if you have additional resources that might be helpful, please reach out to me by email.  About...

...topic page Read Joachim's Series on Choosing the B(right)est Fluorescent Protein Plasmids 101: Visualizing...

...organized by graduate student researchers. That’s right, #ActualLivingScientists, eager to communicate their...

...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...

...plasmids at Addgene, along with other tools like high resolution Ras sensors and crosslinked peptides ...

...and career goals, or to answer any questions you might have about Addgene. The page is divided into three...

...the Expression and Insert Species filters on the right-hand side of the search page to narrow your keyword...

...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...

... University with the backing of a prestigious Fulbright Fellowship. Dr. Kwapata now works as a lecturer...

...plasmids at specific molar ratios. To get these ratios right, you need to do a few calculations. This calculator...

... about Crystal’s exciting career path, to gain insight into industry research, and to get advice on how...

...fluorescent protein family that give off color in ambient light. Expressing these chromoproteins in E. coli would...

...back on some fascinating Addgene history that you might not have known about. Here's the schedule for the...

...finally warming up in Boston and that means summer is right around the corner. Here at Addgene, we’re always...

...winner will be determined by popular vote. That’s right - you get to choose the new name! Additionally, ...

...of the contest is our traditional approach. You might decorate your lab in pipette tip snowflakes, hang...

...shape Addgene's future. Listen to this episode for insights into the process of getting an MD PhD, the inside...

...interneurons. Take a look to see if these new preps might enhance your research!   Table 1: New ready-to-use...

...plasmids shipped. Reaching one million plasmids shared might have you wondering how many plasmids leave Addgene...

...open to industry scientists. This collection is highly diverse in terms of target organisms (from bacteria...