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We narrowed to 986 results for: IGH@;

Showing: 841 - 880 of 986 results

  1. How to Negotiate a Successful Lab Start Up Budget

    Type
    Blog Post
    ...financial aspects. Money often carries an emotional weight. Therefore, too much focus on money can dissuade...point, your budget bottom line costs may be rather high. That’s normal, because this is where you can start...

  2. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    ...components of CGBEs. Their eOPTI-CGBE and cOPTI-CGBE had higher editing efficiency and produced fewer indels than...accessible. It's not entirely clear why this is, but it might point to a more dynamic picture of chromatin than...

  3. CRISPR Library Amplification

    Type
    Protocol
    ...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...

  4. Protocol - How to Inoculate a Bacterial Culture

    Type
    Protocol
    ...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...

  5. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ...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...

  6. Pouring LB Agar Plates

    Type
    Protocol
    ...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...

  7. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ...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....

  8. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...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...

  9. Lentivirus Production

    Type
    Protocol
    ...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 ...

  10. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...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 ...

  11. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ...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...

  12. General Transfection

    Type
    Protocol
    ...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...

  13. Plasmid Cloning by Restriction Enzyme Digest (with Protocols)

    Type
    Protocol
    ...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...

  14. Centrifugation

    Type
    Protocol
    ...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 ...

  15. Protocol - Bacterial Transformation

    Type
    Protocol
    ...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...

  16. AAV Titration by qPCR Using SYBR Green Technology

    Type
    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...

  17. Western Blot

    Type
    Protocol
    ...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...

  20. 'Tis the Season to #DeckTheLab

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

  23. Protocol - How to Purify DNA from an Agarose Gel

    Type
    Protocol
    ...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...

  24. The Twelve Days of Antibodies

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

  25. Protocols for Molecular Biology, Plasmid Cloning, and Viral Preps

    Type
    Protocol
    ...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...

  27. The 12 Days of CRISPR: 2021

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

  28. #Deckthelab Contest Winners 2017

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

  31. Twenty Years of Sharing Science!

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

  38. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    ...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 ...
Showing: 841 - 880 of 986 results