...(Lenti_sgRNA_EFS_GFP) 65656 Mammalian/Lentiviral LIC none S. pyogenes GFP Vakoc U6>sgRNA(F+E) 59986 Other...RNA Targeting RNA Targeting RNA Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled...

... all publications arising from the use of this vector. A.2 Map of pLKO.1 pLKO.1 is a replication-incompetent...terminal repeat. f1 ori f1 bacterial origin of replication. Amp R Ampicillin resistance gene for selection...bacterial cells pUC ori pUC bacterial origin of replication. 5’LTR 5’ long terminal repeat. RRE Rev response...it has been diluted. Mix by swirling or gently flicking the tube. Incubate for 5 minutes at room temperature...the walls of the tube. Mix by swirling or gently flicking the tube. f. Incubate for 20-30 minutes at room... on the size of your target plate. TIP: MOI (multiplicity of infection) refers to the number of infecting...Appendix I.1. Sequence of pLKO.1 TRC-Cloning Vector Click here to see the sequence of pLKO.1 TRC-cloning vector...

...B): LiCl selectively precipitates RNA versus DNA or proteins. Add the correct amount of 7.5 M LiCl solution...solution to bring the concentration of LiCl in the extracted aqueous layer to 2.5 M. Incubate at -20°C ... not affect the quality of RNA or downstream applications. To improve yield of RNA, instead of incubating...

...Load 5 μL of each standard in duplicate Load 5 μL of each sample in duplicate. Do not forget to include a...dilution is appropriate). Quality of duplicates: Exclude duplicates from analyses if there is more than...Considerations Always run standards and samples in duplicate at least If possible, include an AAV reference...standard with DNase ** Make 6 serial dilutions, in duplicate, of your standard curve plasmid (2 x 10 9 stock...

...The amount of DNA that you cut depends on your application. A diagnostic digest typically involves ∼500 ...volume usually varies from 10-50 µL depending on application and is largely determined by the volume of DNA...manufacturer’s instructions. Pro-Tip Depending on the application and the amount of DNA in the reaction, incubation...you will be using the digested DNA for another application (such as a digestion with another enzyme in a...

...bacteria are used as the means for both storing and replicating plasmids. Because of this, nearly all plasmids...expression) carry both a bacterial origin of replication and an antibiotic resistance gene for use as ... microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few ...

...antibody can be purified for use in a variety of applications. Sharing speeds science. We believe that sharing...antibody-expressing plasmids containing the SV40 origin of replication use a HEK293 line stably expressing the SV40 ... containing the Epstein-Barr virus origin of replication use a HEK293 line stable expressing Epstein Barr...

...sufficient quantities of library for experimental applications. Repeated amplifications should be avoided as...required for bacterial propagation (origin of replication and antibiotic selection). This recombination...each 50 µL aliquot of thawed Endura Duos on ice. Flick gently to mix. Electroporate cells (one at a time...

... lentiviral vector allows you to control the multiplicity of infection (MOI) in downstream studies. This... these cells can still be used in downstream applications. Safety Warnings Lentivirus is generally considered...

...lentivirus can be used for a variety of downstream applications such as stable-cell line generation. Workflow...mixture. Add the diluted PEI dropwise while gently flicking the diluted DNA tube. Incubate the mixture 12–...

...amplify and design primers that will bind to and replicate it. The following image shows the ends of the ...mutation than restriction enzyme based cloning. DNA replication by PCR has error rates that range from roughly...

...BSA standard that range from 0–2000 µg/mL . In duplicate, dilute 10 µL of standard, blank, and lysate samples... nm . Calculate the average absorbance of the duplicate samples on the plate. Subtract the average absorbance...

...mini-prepped DNA onto the pedestal. Close the lid and click measure, be sure to record the concentration and...

...of the UV and with a clean, sterile razor blade, slice the desired DNA fragment from the gel. Notes: To...

.... You can find Addgene's sequencing results by clicking on the "View Sequences" link on the Plasmid Information...

...according to speed and time (and temperature, if applicable) listed in your protocol. Pro-Tip Spin speed ...

...DNA. Add the diluted PEI dropwise while gently flicking the diluted DNA tube. Incubate the mixture 15–...

... 3:1 ratio is not working or when doing more complicated cloning. While 3:1 will get you in the ballpark...

...Link opens in a new window) . Gibson Assembly® is licensed to New England Biolabs by TelesisBio. Gibson DG...

...as methanol or acetone may be better for some applications. Remove the paraformaldehyde and follow your...

...lentiviral preparation allows you to control the multiplicity of infection (MOI) in downstream studies. This...

...information on determining DNA concentration and purity click here . Top Protocol: Kit-free Alkaline Lysis Plasmid...

... of a heat resistant DNA polymerase from the thermophilic bacterium, Thermus aquaticus (Taq) . Taq polymerase...

...lower dilutions depending on your downstream applications. If you’ve titered your virus beforehand, you...

...quantification. This protocol was modified from the publication Lock et al. (2014) (Link opens in a new window...

... layers are covered with media. 293T cells are delicate and detach very easily - media should always be...

... sample. This protocol was modified from the publication Wang et al. (2018) . Before Starting Thaw the...

...sensor, cytosolic or ER-targeted eZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic and ...and small molecule indicator: implications for quantification of cytosolic Zn(2+). ACS Chem Biol. 2013 ...:7351. Tetsuya Kitaguchi cAMP (cyclic AMP) Fluorescent sensor of cyclic AMP (cAMPr) for mammalian or insect...sensor for cyclic AMP. Sci Signal. 2018 Mar 6;11(520). pii: 11/520/eaah3738. Justin Blau cAMP (cyclic AMP) ...:4003-4012.e6. Xavier Nicol cyclic di-GMP FRET-based biosensor for cyclic-di-GMP measurement Asymmetrical...Fluorescent biosensor of the cytosolic NADH/NAD+ redox state Imaging Cytosolic NADH-NAD(+) Redox State with...protein for NADH/NAD+ ratio Cytosolic NADH-NAD Redox Visualized in Brain Slices by Two-Photon Fluorescence...

...single assay. Overview of Antibody Applications Antibody-based applications can be generally classed into ...these applications, visit the Antibody section of the Addgene Protocols page. Antibody Applications - Quantification...production and storage techniques, and explain common applications. Science... Handling Visualization Signal Amplification Application Overview Quantification Methods Capture Methods...of other antibodies. This has useful research applications, and is covered in the following Isotypes section...signaling molecules and are often used in clinical applications. Diabodies - Diabodies contain two Fab fragments... molecules that can be distinguished in your application. The indirect detection method uses an unconjugated...

...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Browse CRISPR Plasmids By Function Genome Editing...RNA Targeting RNA Targeting RNA Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Nick CRISPR/Cas nickase mutants introduce gRNA-targeted...Plasmid Gene/Insert Promoter Selectable Marker Publication Prime Edit Cas9 H840A nickase fused to a reverse...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Activate Catalytically dead dCas9 fused to a ...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Interfere Catalytically dead dCas9, or dCas9 ...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Epigenetics To make targeted epigenetic modifications...