...viral delivery RNA delivery of Cas9 and gRNA In vitro transcription reactions generate mature Cas9 mRNA... to target cells through microinjection or electroporation Transient expression of CRISPR components Expression...ribonucleoprotein (RNP) complexes Purified Cas9 protein and in vitro transcribed gRNA are combined to form a Cas9-gRNA...repaired DSBs): For small nucleotide edits that introduce a novel restriction site. The region of interest...identify DNA fragments. PCR amplification and gel electrophoresis (for HDR or NHEJ): For large deletions or insertions...25408407 Hashimoto, M., & Takemoto, T. (2015). Electroporation enables the efficient mRNA delivery into the...enables efficient protein-based genome editing in vitro and in vivo. Nature Biotechnology . 33 (1), 73–80...

...selectively control and investigate cell signaling pathways. Receptors are remotely controlled via small...small molecules, which allows for specific control of activity. Read More CRISPR Class 2 C lustered R egularly...research utilize light to detect, measure, and control molecular signals and cells in order to understand...Lentivirus Adeno-associated Virus (AAV) Adenovirus γ-Retrovirus Read More...

...biosensors, and monitoring protein localization; an introduction to non-protein fluorophores; and much more! ...About This guide to viral vectors contains an introduction to each vector and its applications, tips for...for using vectors in your experiments, troubleshooting advice on production and use, and much more! Close...Edition, trans. About Una traducción al español de nuestro libro electrónico, CRISPR 101. Close About Overlay...Edition About Our newest eBook contains practical introductions and helpful tips for antibody-based applications...

...principle to Gibson cloning, but instead of an in vitro reaction with purified enzymes, it takes advantage... manner. To accomplish this, you just need to introduce into the yeast the two (or more) fragments of ...

...plasmid cloning, and virus, including: Gel Electrophoresis Bacterial Transformation Plasmid Cloning Creating...

... mix. Electroporate cells (one at a time for a total of eight electroporations): Electroporator Conditions...centrifuge BioRad Electroporator (MicroPulser TM , Bio-Rad 1652100) Reagents 200 µL electrocompetent cells (Default... 10 cfu/µg) commercially prepared electrocompetent cells is strongly recommended. 800 ng pooled library...and have 1 mL SOC per electroporation readily available for post-electroporation recovery of cells. Ensure... How do I process my Addgene pooled library? Introduction Please refer to our pooled library material ...according to the actual number. Required Quality Control (QC) Pooled libraries can be challenging and expensive...Stbl4 cells or other ultra-high efficiency electrocompetent cells that are suitable for unstable or recombination-prone...

...single channel pipette Pipette controller Microcentrifuge Electrophoresis chamber Power supply Rocking ... 1mm thick, 10-well, Invitrogen NP0321BOX 20X MOPS SDS running buffer, Invitrogen NP0001 4X NuPage sample...buffer, Invitrogen NP0007 10X NuPage sample reducing agent (500 mM dithiothreitol, DTT), Invitrogen NP0009...Antibodies Recombinant Antibody Purification Protocol Introduction This protocol describes how to determine the...separated by denaturing polyacrylamide gel electrophoresis alongside serial dilutions of a standard antibody...NP0009 PageRuler Plus Prestained protein ladder, Invitrogen 26619 Pipette tips, 10 µL, VWR 76322-132 Pipette...the bottom of the gel. Place the gel in the electrophoresis chamber and secure it. Note: Check the manufacturer...

... Protocols Agarose Gel Electrophoresis Agarose Gel Electrophoresis You may also like... Restriction...DNA from an Agarose Gel DNA Ligation Introduction Gel electrophoresis is the standard lab procedure for ...towards the positive electrode. Always Run to Red. Turn OFF power, disconnect the electrodes from the power ...Standard protocol for performing agarose gel electrophoresis, including tips to improve resolution and separation...pairs) for visualization and purification. Electrophoresis uses an electrical field to move the negatively...through an agarose gel matrix toward a positive electrode. Shorter DNA fragments migrate through the gel...protocol video below to learn how to perform gel electrophoresis. Equipment Casting tray Well combs Voltage ...

...Addgene Plasmid Description pLKO.1 – TRC control Negative control vector containing non-hairpin insert. ...serum-free media Invitrogen: #31985 Dulbecco’s Modified Eagle Medium (DMEM) Invitrogen: #11995 Fetal Bovine...Bovine Serum (FBS) Invitrogen: #16000 Penicillin/Streptomycin Invitrogen: #15140-122 Polypropylene tubes...the TRC for expression of shRNAs. pLKO.1 can be introduced into cells via direct transfection, or can be...subsequent infection of a target cell line. Once introduced, the puromycin resistance marker encoded in pLKO...sites in place of the stuffer. The AgeI site is destroyed in most cases (depending on the target sequence.... pLKO.1 – scramble shRNA Negative control vector containing scrambled shRNA. psPAX2 Packaging plasmid...

...Pipette controller Benchtop centrifuge compatible with 50 mL conical tubes NanoDrop spectrophotometer 37 °...Immunocytochemistry Protocol Western Blot Protocol Introduction This protocol describes how to affinity purify...concentration of each fraction on the NanoDrop Spectrophotometer using the A280 IgG setting. Combine all eluates...concentration of the pooled sample on the NanoDrop Spectrophotometer using the A280 IgG setting. If the concentration...Determine antibody concentration on the NanoDrop Spectrophotometer. Dilute antibody to 1 mg/mL with PBS if needed...to check the concentration on the NanoDrop Spectrophotometer to see if the sample has reached the desired...Periodically check the concentration on the NanoDrop Spectrophotometer to see if the sample has reached the desired...

...of each well at 450 nm on the spectrophotometer. Use the spectrophotometer to generate a standard curve...Antibody Purification Protocol Antibody Guide Introduction This protocol describes how to run an indirect...antibody incubation and plate read Tips and Troubleshooting The optimal concentration of primary antibody...an automated ELISA plate washer. Equipment Spectrophotometer compatible with 96-well plates 1–10 µL single...Multichannel pipette reagent reservoirs Pipette controller Microcentrifuge Microplate shaker ELISA plate... antigen Purified recombinant antibody Isotype control antibody HRP-conjugated isotype-specific secondary...mix. Dilute the primary antibody and an isotype control antibody to the desired concentrations in the antibody...

[email protected] with any questions. Intro to the Lab Bench Introductory techniques designed to help you get...Antibodies Protocols for common antibody applications. Intro to the Lab Bench Name Description (Link opens in...Quantification Measure DNA concentration with a spectrophotometer Restriction Digests Cut and analyze plasmid...plasmid DNA with restriction enzymes Agarose Gel Electrophoresis Separate DNA by size on an agarose gel Watch...DNA using DNA ligase Bacterial Transformation Introduce DNA into a bacterial strain Watch the Video! CRISPR...Description Link to Video General Transfection Introduce plasmid DNA to mammalian cells Lentivirus Production...Antibodies Name Description Link to Video Transfection Introduce plasmid DNA to mammalian cells to produce antibodies...

...DNA Ligation Introduction Transformation is the process by which foreign DNA is introduced into a cell....should also add a positive control (many companies include a positive control plasmid with their competent...ligation of inserts to vectors than for an intact control plasmid. Incubate the competent cell/DNA mixture...transform large plasmids, it is a good idea to use electro-competent cells. Instead of relying on the heat-shock...heat-shock to cause the cells to take up the DNA, an electro-magnetic field is applied to the cell/DNA mixture... do this you will need to have access to an electroporator and the appropriate cuvettes. Follow the manufacturer's...

...and FAQ Do controls When doing ligations you should ALWAYS do a vector alone + ligase control. This will...ligation. Additional controls are encouraged, but may only be required for troubleshooting failed ligations...The following table indicates the various controls: Control Ligase Interpretation Uncut vector - Checks...know has only been thawed once before. Always do controls. See Tips and FAQ below for details. Try different...treated, that the phosphatase treatment worked. This control should, in principle, be free of colonies, but ...

...purity. This can be done with a spectrophotometer. A spectrophotometer uses the absorbance/transmission... instructions for the spectrophotometer specific to your lab. Spectrophotometer Tips Before measuring ...labs have a NanoDrop, which is a very small spectrophotometer that can accurately read DNA concentration...measuring any samples, be sure to ‘blank’ the spectrophotometer using the solution the DNA is resuspended in, ...NanoDrop. It is a good idea to re-zero any spectrophotometer between samples if they are expected to vary...

...an uncut sample of each vector to help with troubleshooting if your digests don’t look as you expected....product is the anticipated size and that your band is strong (indicating that the PCR reaction worked and that...insert. It is also important to set up negative controls in parallel. For instance, a ligation of the recipient...much less total DNA (<1ng) or if you are having trouble getting colonies, you might want to use higher ...to be very large (>10kb) you might want to use electro-competent cells instead of the more common chemically-competent...plasmid alone plate. The recipient plasmid alone control will tell you your “background” level or more specifically...see any colonies, you should conduct a positive control to ensure that your transformation worked. You ...

...Restriction Digest of Plasmid DNA Agarose Gel Electrophoresis Introduction Restriction enzymes are naturally occurring...and analyze the resulting fragments by gel electrophoresis . The pattern of the fragments on the gel can...to analyze a restriction digest. Equipment Electrophoresis chamber Pipetman Pipet tips Reagents Liquid...manufacturer's instructions) Gel loading dye Electrophoresis buffer Verifying Total Plasmid Size -OR- Insert...avoided. If you do have to do so, there is no way to control which orientation the insert is ligated into the... tricks see the Restriction Digest and Gel Electrophoresis pages....

... uncut sample of each plasmid to help with troubleshooting if your digests don’t look as you expected....insert. It is also important to set up negative controls in parallel. For instance, a ligation of the recipient...much less total DNA (<1ng) or if you are having trouble getting colonies, you might want to use higher ...to be very large (>10kb) you might want to use electro-competent cells instead of the more common chemically-competent...plasmid alone plate. The recipient plasmid alone control will tell you your “background” level or more specifically...see any colonies, you should conduct a positive control to ensure that your transformation worked. You ...depending on the number of background colonies on your control plate (the more background, the more colonies you...

...channel pipette Pipette controller Pipette tips and pipettes Spectrophotometer Heat block Mini gel tank...Protocol Recombinant Antibody Purification Protocol Introduction Western blot is a technique used to separate...ladder. Place the lid on the tank and plug the electrode cords into the power supply. Run the gel at 100... a dark room to detect the bands. Tips and Troubleshooting The optimal lysis buffer will vary depending...as expected and is specific, include a positive control sample that you know expresses the protein, such...transfected with the protein of interest, and a negative control sample that does not express the protein of interest...

...Viral Service Lentivirus Guide Virus Blog Posts Introduction This protocol describes how to use droplet digital...ribonuclease P/MRP 30 kDa subunit (RPP30) are used as a control for normalization. The dilution series outlined...wells containing the virus and the untransduced control. Pro-Tip For even seeding, prepare a batch for ...Determine the concentration of each sample on a spectrophotometer. Prepare 25 ng/µL stocks of each sample. Samples...calculate the final infectious titer. Tips and Troubleshooting We recommend wiping down all pipettes and equipment...concentration of RRE positive droplets in the untransduced control should be close to zero (A01). In this protocol...