...2):a001107. PMC PMID: 20182618 . Do you have suggestions for other plasmids that should be added to this...

...Ampicillin none, need Cas9 plasmid Qi Do you have suggestions for other plasmids that should be added to this...

...SCARF-bio-His 51827 SEMA4-bio-His 51829 SPN-bio-His 51830 STIM1-bio-His 51831 TM9-bio-His 51832 TMED1-bio-His 51852...

...and envelope used. Please consult with your institution’s Biosafety Committee before beginning virus work...

... of the primer assist with restriction enzyme digestion (usually 3-6bp). Restriction Site: Your chosen...and then TAAGCA to improve restriction enzyme digestion. This gives us a sequence of 5'-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA... You can generate the reverse-complement using existing software (a quick internet search will lead you... The PCR product is now ready for restriction digestion. Digest Your DNA Set up restriction digests for...have compatible overhangs or no overhangs after digestion, you will need to use a phosphatase to prevent...colonies are a result of uncut empty plasmid, you will still have colonies when you do not add ligase. If the...purification. After purifying the DNA , conduct a diagnostic restriction digest of 100-300ng of your purified...

...variety of Cas-containing plasmids. Do you have suggestions for other plasmids that should be added to this...

...yes, cut S. pyogenes Neomycin Chen Do you have suggestions for other plasmids that should be added to this...

...called "sticky ends", are what allow the vector and insert to bind to each other. When the sticky ends ...depicts the ligation of two sticky ends that were generated by EcoRI digestion: Usually, scientists select...that have been generated by restriction enzyme digestion . Most restriction enzymes digest DNA asymmetrically...ligating to itself during the ligation process. If the sticky ends on either side of the vector are compatible...

...molecules such as TAL proteins and the CRISPR system consisting of a catalytically inactive form of Cas9 (dCas9...

...with serial dilutions of the lentiviral vector, harvesting cells several days later, and isolating genomic...

...Protocol Diagnostic Restriction Digest Verify a plasmid using restriction enzymes Diagnostic Restriction...free to email us at [email protected] with any questions. Protocols Video protocol guides for standard ...- Requests Your guide to the MTA process when requesting samples from Addgene Technology Transfer How ...Ordering Page MTA FAQs Answers to frequently asked questions about the MTA process Help Center How to Deposit...

... with the recipient plasmid's MCS. However, you still need to avoid restriction enzymes that cut within...have compatible overhangs or no overhangs after digestion, you will need to use a phosphatase to prevent...colonies are a result of uncut empty plasmid, you will still have colonies when you do not add ligase. If the...purification. After purifying the DNA , conduct a diagnostic restriction digest of 100-300ng of your purified...orientation of your insert, so you may want to design a diagnostic digest for this purpose. Congratulations, you...

...gel from the plastic wrapper and rinse with deionized water. Gently remove the white sticker on the bottom...Rocking platform Fume hood Metal spatula Razor blade Plastic tray Gel imaging system ImageJ or similar software...Section 2: Staining the Gel Place the gel in a plastic tray with 100 mL of deionized water. Rinse gel ...the standard curve is y = 14725x + 3748.4, then substitute 18179.442 for y and solve for x. 18179.442 = ...

...follows: Remove the gel from the plastic packaging. Remove tape and plastic comb. Rinse the wells with deionized...well microtiter plate Chemiluminescence substrate Plastic wrap Primary antibody Secondary antibody Deionized... white separator. Keep the Bottom Stack in the plastic tray. Place the Bottom Stack on the blotting surface...for 5 min at RT . Cover the membrane with clear plastic and use a gel imager with chemiluminescence detection...

...molecular biology, plasmid cloning, and titering and testing your viral preparations. ...free to email us at [email protected] with any questions. Intro to the Lab Bench Introductory techniques...your plasmids. Virus Protocols for titering and testing your virus preparations. Antibodies Protocols for...Amplification Amplify CRISPR pooled-plasmid libraries Diagnostic Restriction Digest Verify a plasmid using restriction...

... Gibson and colleagues at the J. Craig Venter Institute developed a novel method for the easy assembly...the assembled parts. Tips and Troubleshooting “Stitching” fragments together using oligos When you need...sequence between two PCR products, one method is to “stitch” together several oligos. This technique is especially...Pro-Tip Please note that the way to design the “stitching” primers and the amounts of primers to include...

...digital PCR, in vitro and (when possible) in vivo testing, and full sequencing of the final viral vector ...

... Type Backbone Mutations Tags PI Do you have suggestions for other plasmids that should be added to this...

...controller Hazardous waste container pH meter Stir plate Magnetic Stir Bar Reagents DMEM high glucose, Corning... of powder in 100 mL of deionized water. While stirring, slowly add hydrochloric acid until the solution...any packaging cells that were collected during harvesting. Filter supernatant through a 0.45 μm PES filter...

...allows you to distinguish objects within the sample. You can increase contrast by a) adjusting the aperture.... You can take entire courses on microscopy and still have more to learn, so, for this protocol, we'll...image larger) and resolution (the ability to distinguish between two discrete objects). Figure 1 depicts...