... 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...