...Syn-driven, Cre-dependent Cre-dependent expression of membrane-bound GFP and synaptophysin-mRuby for labeling...

...start getting results from your AAV experiments, remember to share your protocols and results through Addgene...

...brain in mice and see if they are better able to remember how to solve a maze. These engineered viral genomes...

...Vector Addgene Plasmid 10878. Protocol Version 1.0. December 2006. Copyright Addgene 2006, All Rights Reserved... . 1996 272:263-267. PubMed . Asymmetry in the assembly of the RNAi enzyme complex. . Schwarz DS et. al...

...Enzyme parts have C/E overhangs for Golden Gate assembly. Plasmids Explore the collection and order individual...

...filtration through a 0.45 µm polyethersulfone (PES) membrane. The preparations are then aliquoted, frozen, ...

...against these foreign nucleic acids. Acr family members inhibit CRISPR by a variety of mechanisms, including...Mediated Genetic Screening in Mice with Haploid Embryonic Stem Cells Carrying a Guide RNA Library. Cell ...

... use in SapTrap reactions for targeting vector assembly, and the remaining 5 plasmids include Cre and ...

...Lines pSpCas9(BB) (pX330) Link Parrott NEBuilder Assembly of CRISPR vectors using ssDNA oligos p201G Cas9...

... your membrane, run a double-membrane transfer with a psi membrane stacked behind the p membrane. If your...through the p membrane into the psi membrane, that’s a good indication that the p membrane pores are too...marked on the membrane (1) using the gel as a guide. The gel is then removed from the membrane (2) and the...troubleshoot.   Before you start… Mark your membrane! Membranes look the same from both sides, so it’s quite...appropriately sensitive. If using a PVDF membrane, you can dry the membrane, wet it with 20% methanol, and place...completely through the membrane or larger proteins not transferring completely onto the membrane. If you’re not...completely through your membrane to the other side), you can check by using two membranes during your transfer...

... your gel and membrane transfer as normal. Immediately after transfer, dry the membrane to maximize protein...manufacturer’s instructions for the membrane you are using. Dry nitrocellulose membranes are brittle, so proceed ... as normal! Stripping your membrane Alright, so you’ve dried your membrane and returned it to buffer, ...depend on your specific experiment. Remember, stripping your membrane will remove some of your sample —and...’ll want to: Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...a fume hood. Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...you need to start a whole new blot? Never fear: membrane stripping is here! Rather than start over, you...

...the RSRT along with individual laboratories to assemble a Rett Syndrome plasmid resource for the scientific...

...off-target editing sites in the embryos, as well as whole exome sequencing of embryo derived-ES cells.They also...editing between iPS cells and embryos make a case for conducting research in embryos should our society decide...sequencing to a small percentage of edited embryos. Since the embryos produced were not being used to establish...-free embryos to only 60%? Unfortunately, given the differences between CRISPR repair in embryos and iPS...this paper. Any hint of CRISPR editing in human embryos has been met with a storm of media coverage. But... replication. Indeed, they observed that 41/42 embryos were non-mosaic at 3 days post fertilization. Since...MYBPC3 allele. Interestingly, they found that the embryos underwent repair not from the single-stranded oligonucleotide...

...nitrocellulose membranes and exposed to IPTG in order to induce protein expression. Cells on the membranes will ...orientation of each membrane on its plate before moving on. Then peel each membrane off the plate (saving...high salt.  To probe membranes with target bacterial cells, wash your membranes with PBST to remove salt... on the membrane to bind their target cells, pulling them out of solution. Wash your membranes well with... 20 Petri dish-sized circles of nitrocellulose membrane and press each gently down onto a plate of colonies...regrow the lifted colonies overnight), flip the membrane over and place it colony-side up onto a fresh ...plasmid selection. Remove air bubbles between the membrane and the agar using forceps. Several protein induction...

...multicomponent assembly, as fragments with overlapping sequence homology can be assembled in a unique order...multicomponent assembly have 5′ or 3′ sequence homology to each other, they may be assembled incorrectly.... one option is to perform a hierarchical assembly, assembling fragments in multiple steps to avoid using...Golden Gate assembly, may also be beneficial. SLIC is most often compared to Gibson assembly, a cloning...required for Gibson assembly. SLIC is a standardized method for multi-fragment DNA assembly, and its low cost... by Mary Gearing in December 2015 and was updated by Emily P. Bentley in September 2024.  References ... cheap, standardized, and rapid multi-part DNA assembly method. Read on to learn how to use it in your...

...to use some assembly methods such as Golden Gate or Gibson assembly. Golden Gate Assembly of gRNAs Golden...Golden Gate/Gibson Assembly Multiplexing Plasmids: These plasmids allow you to assemble 2-4 gRNAs through...gRNAs. PTGs are assembled into a wt Cas9-containing vector using Golden Gate assembly, and up to 8 gRNAs...Multiplex Plasmid: Two gRNAs can be assembled using Gibson Assembly or SLIC cloning. gRNAs are expressed...Bullock lab, and gRNAs can be inserted using Gibson Assembly or SLIC cloning methods. A BsaI-based E. coli ... multiplexed gRNAs with Golden Gate and Gibson assembly methods If you want to scale up the number of ...Golden Gate Assembly uses Type IIS restriction enzymes, which cleave outside of their recognition sequence...

...can be used to assemble multiple fragments - up to 10 fragments are commonly assembled in a single reaction... Gibson assembly require 20-40 bp of homology at the ends of DNA fragments to specify assembly order, ...sequence homology cannot be assembled using this method, but can be assembled with Golden Gate. The popular...acids, but Golden Gate assembly can be designed to be scarless. Golden Gate assembly is also less expensive...recognition sequence, they can be customized to direct assembly of DNA fragments. When designed correctly, the...sites with complementary overhangs that direct assembly of the final ligation product. As shown below,...higher number of potential clones. Golden Gate assembly has a few advantages over other cloning methods...

...organization scheme for multi-part DNA assembly. Simply put, it assembles individual DNA components into larger...MoClo-compatible vectors. Level 1: Assembly of multiple Level 0 parts into TUs. Level 2: Assembly of multiple Level 1...IIS restriction enzyme and a ligase are used to assemble multiple DNA fragments together in a one-tube ... Fig 1. MoClo enables individual parts to be assembled into larger functional units which are transcribed... Understanding how individual parts can be assembled into TUs is key to understanding MoClo. By a similar...Construction of multigene constructs, through multi-TU assembly,happens via three steps: Level 0: Cloning of ...multigene constructs. Figure 2: The three assembly levels of MoClo. Image courtesy of Rob Hurt. ...

...off-target effects Can be used to generate transgenic embryos Cas9-gRNA ribonucleoprotein (RNP) complexes Purified...

... pAAV-S5E2-GFP-fGFP E2 regulatory element GFP, membrane-targeted GFP Control Dimidschstein 192552 pAAV-CAG...