... tank. Pro-Tip Different SDS-PAGE gel tanks will have different gel orientations. Refer to the instructions...protocol uses a dry transfer device but can be adapted for wet and semi-dry transfer approaches. Last Update...80 °C freezer Reagents 1X PBS Lysis buffer e.g., RIPA lysis buffer Microcentrifuge tubes BCA Assay, Thermo...β-mercaptoethanol 4X protein loading buffer Precast SDS-PAGE gel SDS-PAGE running buffer Prestained protein ladder...appropriate volume of cold lysis buffer. Pro-Tips The volume of lysis buffer will vary depending on the size...lysis buffer will vary depending on the cellular location of the protein of interest. RIPA buffer is suitable...β-mercaptoethanol to the 4X protein loading buffer. Dilute 4X protein loading buffer in the sample to 1X . Boil the...

.../G binding buffer (20 mM sodium phosphate buffer, pH 7.0) 20 mL 1M sodium phosphate buffer 980 mL of deionized...use. Warm the affinity column, binding buffer, and elution buffer to room temperature (RT) before use. ... pour off the ethanol storage buffer. Tightly attach LabMate PD10 Buffer Reservoirs to the top of the ...to Option 2 with a buffer exchange/concentration using an Amicon 30 kDa MWCO buffer exchange column. (...may need to be adjusted to accommodate slight differences between products. Addgene does not endorse or...Timeline Day 1: Purify antibody Day 2 or later: Buffer exchange Equipment Class II, Type A2 Biological...Filter, 0.2 µm (luer-lock), VWR 431229 IgG Elution Buffer, Thermo Fisher 21028 Protein G GraviTrap columns...

...there is very little difference between the two. Note: Make sure to use the same buffer as the one in the...the gel box (do not mix different buffers and do not use water). Microwave for 1-3 min until the agarose...the running buffer when you run the gel. If you do not add EtBr to the gel and running buffer, you will ...Agarose Gel: Add loading buffer to each of your DNA samples. Note: Loading buffer serves two purposes: 1...bubbles or buffer from entering the tip. Place the very top of the tip of the pipette into the buffer just ...the gel and buffer, place the gel into a container filled with 100 mL of TAE running buffer and 5 μL of...but do not overboil the solution, as some of the buffer will evaporate and thus alter the final percentage...

... NEB buffer 1 NEB #B7001S NEB buffer 2 NEB #B7002S T4 DNA ligase NEB #M0202S T4 DNA ligase buffer NEB ...lentiviral infection and selection G. Safety H. References H.1 Published articles H.2 Web resources I. Appendix...original pLKO.1-TRC cloning vector has a 1.9kb stuffer that is released by digestion with AgeI and EcoRI... into the AgeI and EcoRI sites in place of the stuffer. The AgeI site is destroyed in most cases (depending... a complete map of pLKO.1 containing the 1.9kb stuffer, visit www.addgene.org/10878/ . Description Vector...than others. In addition, demonstrating that two different shRNAs that target the same gene can produce the...The pLKO.1-TRC cloning vector contains a 1.9kb stuffer that is released upon digestion with EcoRI and ...

... Prepare 1X dilution buffer (see recipe above). Save ~25 µL of 1X dilution buffer in a microcentrifuge...5 µL in 95 µL 1X PCR buffer (1:20) Dilution 2 (20X): 5 µL in 95 µL 1X PCR buffer (1:400) Dilution 3 (20X... µL in 95 µL 1X PCR buffer (1:8,000) Dilution 4 (20X): 5 µL in 95 µL 1X PCR buffer (1:160,000) Dilution...µL in 95 µL 1X PCR buffer (1:3,200,000) Dilution 6 (2X): 50 µL in 50 µL 1X PCR buffer (1:6,400,000) Dilution... in 50 µL 1X PCR buffer (1:12,800,000) Dilution 8 (2X): 50 µL in 50 µL 1X PCR buffer (1:25,600,000) Use...Vector Guides Virus Blog Posts Molecular Biology Reference Introduction This protocol describes how to use...Grade Water, Hyclone, SH30538.02 GeneAmp 10X PCR Buffer, Applied Biosystems, N8080129 ddPCR Supermix for...

...final wash, aspirate the wash buffer from the wells. Prepare the blocking buffer (1% BSA in PBS) as follows...wash, aspirate the wash buffer from the wells. Prepare antibody dilution buffer (0.1% BSA in PBS) as follows...may need to be adjusted to accommodate slight differences between products. Addgene does not endorse or...temperature or 37 °C. The protocol notes when there are different incubation options available. Caution Use the ...and vortex. Pro-Tip You may need to try a few different concentrations of antigen to determine the ideal...into the first three wells of each row, using a different standard solution for each row of a 96-well ELISA...C . Section 2: Block the plate Prepare the wash buffer (0.05% Tween-20 in PBS) as follows: Pipette 0.5...

...If possible, include an AAV reference sample of known titer. The reference material should have a titer... DNase buffer + 1 μL DNase Gently mix sample (do not vortex) Incubate 30 min at 37 °C Transfer to ice ...an internal reference virus that is 1 x 10 13 GC/mL. We recommend always using a reference within 1-log...should observe differences in Ct values that make sense for your dilutions (~3.3 difference Ct for a 10-...This protocol was validated using an internal reference AAV of known titer, 100837-AAV1, and by measuring...plasmid for standard curve RNase-free DNase 10X DNase buffer Nuclease-free water Microcentrifuge tubes 96-well...master mix should also contain an internal passive reference (typically ROX dye), to normalize non-PCR–related...

...5 mL of Buffer A to 45 mL PBS 0.001% Pluronic-F68 in PBS + 200 mM NaCl (C) (formulation buffer) Add 5 ...NaCl/PBS-MK buffer 25% iodixanol step: mix 5 mL of 60% iodixanol and 7 mL of 1X PBS-MK buffer and 30 μL ...purified virus. Nonetheless, we recommend performing a buffer exchange before using the purified AAV in vivo ...purification. Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note: Both steps could... edge needles, Hamilton 1X PBS pH 7.4 1X PBS-MK buffer 100X Pluronic-F68 NaCl MgCl 2 KCl Centrifugal filter...MWCO 100 kDa) Reagent Preparation 1 M NaCl/PBS-MK buffer Dissolve 5.84 g of NaCl, 26.3 mg of MgCl 2 and ...through a 0.22 μm filter and store at 4 °C. 1X PBS-MK buffer Dissolve 26.3 mg of MgCl 2 , and 14.91 mg of KCl...

... Permeabilization buffer: Dilute 20 µL of Triton X-100 in 10 mL PBS. Blocking buffer: Dilute 0.5 g BSA...platform in 500 µL permeabilization buffer. Remove the permeabilization buffer and dispose of it in an appropriate...rocking platform in 500 µL blocking buffer. Remove the blocking buffer and dispose of it in an appropriate...method. The protocol may need to be optimized for different cells, target proteins, etc. Sharing speeds science...may need to be adjusted to accommodate slight differences between products. Addgene does not endorse or...conical tubes 50 mL conical tubes Before Starting Refer to the manufacturer's instructions for additional...µL Triton X-100 in 10 mL PBS. Antibody dilution buffer: Dilute 0.5 g BSA and 150 µL Triton X-100 in 50...

...Vector Guides Virus Blog Posts Molecular Biology Reference Introduction This protocol can be used to produce...1272 cm 2 Heat-inactivated FBS (HI-FBS) Pro-Tip Different brands and lots of FBS can promote or inhibit ...Polyethylene Glycol 8000 (PEG) + 0.5 M NaCl Cell lysis buffer (50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl2) Reagent...into phases. Aliquot and store at 4 °C. Cell Lysis Buffer : 50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl 2 Add...cells, add 200 mL of DMEM complete media, then transfer the cells into a sterile bottle. Rinse the CS2...RepCap 7,265 1.00 727.6 pHelper 11,854 1.00 1,185 Transfer Plasmid 5,842 1.00 584.2 Total bp 24,961 In total...Volume Needed: 949.8 μg / 1.0 μg/μL = 949.8 μL Transfer Plasmid: 0.08 μg/bp × 5,842 bp = 468.1 μg Volume...

... DNA Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer) 0.5-1μL T4 ...two different enzymes for adding an insert into a vector (one enzyme on the 5' end and a different enzyme... low such that you cannot get all 100ng of DNA, buffer and ligase into a 10μL reaction, scale the reaction...necessary - being sure to increase the amount of buffer proportionally. 1μL of ligase should be sufficient...sufficient for larger ligation reactions. Because ligase buffer contains ATP, which degrades upon freeze/thaw cycles...controls. See Tips and FAQ below for details. Try different vector to insert ratios to optimize the ligation...genes and vectors, this ratio is really meant to refer to the molarity of DNA ends available for ligation...

...When these reagents are combined in an appropriate buffer, a series of heating (denaturing) and cooling (...Bucket 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 mM each nt) 2.5 μL...: 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 mM each nt) 2.5 μL...for pipetting error. In this example, we have 7 different reactions (7 unique primer pairs), so we multiply...single 1.5mL tube combine the following: 10X Taq buffer with MgCl 2 : 5μl x 7 reactions = 35μl total + ...sequence. FAQ How do I design primers? See our reference page on how to design primers for detailed instructions...2 acts to supplement the amount supplied by the buffer stock, where it is known to form a gradient when...

...restriction digest buffer (see manufacturer's instructions) Gel loading dye Electrophoresis buffer Pipet tips ...such as a digestion with another enzyme in a different buffer), but will not be gel purifying it, you may... at specific sequences. There are hundreds of different restriction enzymes, allowing scientists to target...Sequence Analyzer . Determine an appropriate reaction buffer by reading the instructions for your enzyme. Pro-Tip...same time), you will need to determine the best buffer that works for both of your enzymes. Most companies...combine the following: DNA Restriction Enzyme(s) Buffer BSA (if recommended by manufacturer) dH 2 O up ...µg DNA 1 µL of each Restriction Enzyme 3 µL 10x Buffer 3 µL 10x BSA (if recommended) x µL dH 2 O (to bring...

...days. Note that growth rate differences can be inferred by the differences in colony size. Figure 2: Cas9... volume of homogenized cell solution is transferred. Transfer cells from the homogenized cell solution...990 µL complete medium. Instead of transferring 0.125 µL, now transfer 100 times that volume, which is 12.5... solution, transfer 12.5 µL of the 1:100 dilution to 10 mL of conditioned medium. Transfer 100 µL of the...increased serum concentrations, may be optimal for different cell types. Before starting this procedure, be...before counting the cells. When you ultimately transfer approximately 50–100 cells to the conditioned ...

...Collection Antibodies Blog Posts Molecular Biology Reference General Transfection Protocol Recombinant Antibody...may need to be adjusted to accommodate slight differences between products. Addgene does not endorse or...cell density and viability: Using a 5 mL pipette, transfer 0.5 mL of HEK293 cell suspension into a clean ...Gently swirl the flask 5–10 times before sampling. Transfer 10 µL of trypan blue into a clean microcentrifuge...microcentrifuge tube. Vortex the cell suspension. Transfer 10 µL of cell suspension into the microfuge tube containing.... Transfect the flask containing 108 mL cells: Transfer 6 mL of BCD TFX into each of two 50 mL tubes. ...tubes and incubate for 1 h in the 37 °C bead bath. Transfer the PEI-MAX and recombinant antibody plasmid DNA...

...liquid or separate liquids of different densities is essential to many different experimental procedures, ...likely encounter several different types of centrifuges that can accommodate different sized containers, spin... selecting and using a centrifuge to separate different components in a liquid sample. Protocols...piece of laboratory equipment used to separate different components in a liquid sample. It achieves this...spin at different speeds, or keep samples at specific temperatures. This protocol will cover the general...rotor. How to Centrifuge Now that you know the different parts of a centrifuge, you’re ready to begin using...Conclusion Centrifuges are important tools for many different protocols. Improper use of a centrifuge can damage...

...NP0321BOX 20X MOPS SDS running buffer, Invitrogen NP0001 4X NuPage sample buffer, Invitrogen NP0007 10X NuPage...to total 13 µL . Prepare 1X MOPS buffer Dilute 25 mL of 20X MOPS buffer into 475 mL of deionized water....with 200 µL 1X MOPS running buffer. Fill the chamber with 1X MOPS running buffer. Make sure that the chamber...may need to be adjusted to accommodate slight differences between products. Addgene does not endorse or....125 mg/mL: 10 µL of 0.25 mg/mL with 10 µL PBS Transfer 5 µL of each standard to a microfuge tube. Add... antibody to a concentration of 0.9–1.1 mg/mL. Transfer 5 µL of recombinant antibody to a microfuge tube...Procedure Section 1: SDS-PAGE Add 5 µL of 4X sample buffer to each sample. Add 2 µL 10X reducing agent to ...

...This is included in the resuspension buffer of most kits. Reference Page Top Index...bacteria transformed with your plasmid Resuspension buffer Denaturing solution Renaturing solution 2 mg/mL...ethanol or isopropanol 90% ethanol 70% ethanol TE buffer 3 M Na-acetate (pH 4.8) Protocol: Generalized DNA...Grow an overnight culture of bacteria . Pro-Tip Refer to appropriate DNA prep protocol for volume of bacteria...Remove the supernatant and resuspend the bacteria in buffer. Note: This step gets all of the bacteria back ...into suspension, but within a smaller volume of buffer that is compatible with the next solution. Add ... DNA off of the column using water or a neutral buffer such as TE. You will now have plasmid DNA that ...

...samples have significantly different concentrations, it is possible that the difference between them has affected...and consistent readings from DNA in a buffered solution. Reference Page Top Index...Plasmid DNA Background Information During several different stages of molecular cloning, it is important to...particular substance in that liquid. Molecules absorb different wavelengths of light to varying degrees and most...concentration readings than a DNA sample dissolved in buffer (such as TE). You will get much more accurate and...

...restriction digest buffer (see manufacturer's instructions) Gel loading dye Electrophoresis buffer Verifying Total...below we want to know how to differentiate between two clones that differ only in the orientation of the...accurately sized on a gel and also such that they are different enough in size to be easily resolved from each...approach, it is often a good idea to choose two different enzymes that will give you unique but distinct...the expected products from each orientation are different sizes. Simply run the digest products on a gel...

...Molecular Biology Reference How do I process my Addgene pooled library? Introduction Please refer to our pooled... pooled plasmids differ by only a short region of DNA: guide RNAs targeting different genes in an organism's...Picogreen, or Qubit. Each different DNA measurement type can report slightly different concentrations of DNA... Pooled libraries contain tens to millions of different plasmids in a single sample. They are often used...composition of the library. Bottlenecks, fitness differences, and plasmid recombination can all impact the...

... pipette 10–20 times. Use a new pipette tip to transfer 200 µL of the first dilution into the next dilution...min. Resuspend cells in 500 µL DMEM complete and transfer to a microcentrifuge tube. Centrifuge for 5 min...the DG8 gasket, making sure that it is secure. Transfer the cartridge holder to the droplet generator....while slowly and gently pipetting the droplets. Transfer the droplets to a prechilled PCR plate. Pro-Tip... 98 10 2 1 Hold 4 ∞ 2 1 After PCR is complete, transfer the plate to the Droplet Reader. Open the QuantaSoft...genome: Since the cellular gene RPP30 is used as a reference to the cellular genome, it is assumed that there... cells per well is 300,000 and the virus volume refers to the total volume the cells were transduced, ...

...Lentiviral Packaging plasmid(s), Envelope plasmid, and Transfer plasmid encoding your insert of interest Reagent...and 5 mL of glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote or inhibit ...1.3 pmol Envelope Plasmid ( pMD2.G ) 0.72 pmol Transfer Plasmid* 1.64 pmol OptiPro SFM to total volume...concentrations and ratios should be optimized for each transfer plasmid. Pro-Tip Endotoxins can inhibit transfection...PEI is 1:3 (1000 μL total per 10 cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid...gives the highest percentage of GFP positive cells. Refer to the table below for a possible range of ratios...individual harvests are pooled. If pooling harvests, transfer the harvested media to a polypropylene storage...

...Tips Although there are many different types of pipettes that have different mechanisms to disperse liquids...that the same pipette can be used for measuring different samples without cross contamination as long as...performs the same function, the numbers are read differently on each type of pipette. This section will help...Procedure How to Pipette Now that you know the different parts of the pipette, how to select tips, and ...pipette tip. Liquids of various viscosity have different flow rates. The more viscous a liquid, the lower... are pipetting the same amount of liquid into different tubes or into wells of a microplate, you may consider...

...from the final product (no "cloning scars"). A “stuffer” sequence allows for electrophoretic separation...template sequence. 5' and 3' primers will have different leader sequences, but operate on the same principle...at two points by the enzyme BsaI, removing the stuffer sequence and sacB counter-selection marker (see... PCR products with sterile water (instead of TE buffer) to ensure optimal salt concentrations in subsequent... minutes. Volume (μl) Reagent Final Conc. 4 10X Buffer 1X 20-30 Eluted DNA 10-50 ng/μl 1 dGTP (100mM) ...your PCR product before proceeding, as they will interfere with the exonuclease activity of T4 Pol in the...

...restriction enzyme buffers). If you select enzymes that can function in the same buffer, it will save you... to use, you can check out our Empty Backbone Reference . Background Subcloning by restriction digest ... a fusion protein) Ideally, you will find two different restriction enzymes for your subcloning. It is...enzymes will work in the same restriction enzyme buffer (see (Link opens in a new window) New England Biolabs...expression vector and can begin your studies. Reference Page Top Index...

... This approach can be adapted for different cell lines and different transfection reagents. Workflow Timeline...5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote or inhibit ...gives the highest percentage of GFP positive cells. Refer to the table below for a possible range of ratios...Incubate the mixture 15–20 min at RT. Carefully transfer the transfection mix to your HEK293T cells. Add...

...reaction mix) consists of three different enzymes within a single buffer mixture and an optional SSB protein...isothermal reaction. With the activities of three different enzymes, the product of a Gibson Assembly is a...primers to include in the Gibson reaction are different than with normal PCR primers (Gibson et al. , ... than five fragments at a time. Resources and References New England Biolabs (NEB) (Link opens in a new...

... 5mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and FBS lots can promote or inhibit transfection...efficiency of your transduction, you will see different degrees of cell death upon antibiotic selection...polyclonal populations of resistant cells start proliferating and the individual wells become confluent, expand... meaning that the transgene has integrated in different locations in the various cells in the culture....varying expression levels of the transgene from different cells in the population. Once the polyclonal populations...

...Purification Agarose Gel Purification Molecular Biology Reference Introduction This protocol describes how to extract...ThermoFisher Scientific . Figure 1: A diagram of the different steps in RNA extraction. Before Starting RNA is...cells. Extract RNA from the homogenized sample(s).Transfer tissue/cell lysate to a 4 mL tube. Add the following...the cold room may damage it. Using a pipettor, transfer the top, aqueous phase to a new RNAse-free tube...

...shRNAs to any vector (the procedure will simply differ in terms of primer design). Let's assume that your...oligos The oligos should be resuspended in annealing buffer (10mM Tris, pH7.5-8.0, 50mM NaCL, 1mM EDTA) and...total volume of 50μL - add additional annealing buffer if necessary to get to 50μL. Efficient annealing...mini-prepping and verify insert by sequencing. Reference Page Top Index...

...Introduction For many experiments, you’ll need to make buffers, media, or other solutions. A key part of this ...cross-contamination with other substances. They also help you transfer the material to your tube or container. Pro-Tip...weighed out the correct amount of your reagent, transfer it into your container. Make sure that any spills...the accurate composition of your solutions and buffers. Now that you have your reagents weighed out, you...

...FAQ What is the difference between high copy and low copy plasmids? The copy number refers to the number... one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the bacteria...350 - 400 rpm to obtain a higher cell density. Reference Page Top Index...

... a standard plasmid transformation protocol and offer some tips and tricks to reduce transformation time...when requesting samples from Addgene Technology Transfer How to Order A step-by-step walkthrough of Addgene's...Scientist Jessica Welch to discuss her career path, differences in research culture between the US and Australia... importance of international collaboration, and offers career advice for scientists looking to expand ...

...can be used to titer lentiviral preparations that confer antibiotic resistance....can be used to titer lentivirus preparations that confer antibiotic resistance. Determining the titer of...5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote or inhibit ... the incubator. Incubate for 48–72 h. Pro-Tip Different cells may be more sensitive to lentivirus or to...

...because different components of the specimen will refract and reflect the light. These differences create... of biological research and are found in many different types of laboratories. These tools allow you to...objective performs essentially the same task but has different magnifying powers. When you look at the side of...

... one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the bacteria... with resistance to that antibiotic - usually conferred by a plasmid carrying the antibiotic resistance...x (0.220 L) = 8.14 g pre-mixed LB-agar powder. Transfer the LB-agar powder you’ve measured out into an...molten agar from boiling over in the autoclave. Transfer the sterile water (in our case 220 mL) to the ...

...backbone (See Addgene's Sequencing Primers for reference). Many sequencing cores have a list of common ...recommends using Addgene's sequencing results as a reference for primer design. A good sequencing reaction ...reaction with high background: If you obtain a different sequence than expected and wish to contact Addgene...

... potential accidents such as spills. BSL-2 is different because it includes labs that work with agents...cannot perform your duties. Be sure to try out different sizes to know your fit. It is best to not reuse...

... select transformed cells containing plasmids differing in their resistance genes, as one does not need...Sample Data Selection of E.coli on LB-agar using different concentrations of carbenicillin plated over-agar...