...should be properly disposed of following your institution’s hazardous waste procedures. Last Update: February...February 7, 2022 Protocol Video Watch this instructional video to learn how to affinity purify recombinant...for your columns, refer to the manufacturer’s instructions. Attach the Gravitrap column to a clamp on a...

... also use a nucleotide-based molecular insert (inosine) instead of a regular nucleotide for broader pairing...

...of plasmids in your purified DNA. This protocol explains how to isolate a single bacterial colony by streaking...plasmids or bacteria need to be grown at 30 °C instead of 37 °C. This is often true for large unstable...

...Envelope plasmid, and Transfer plasmid encoding your insert of interest Reagent Preparation DMEM Complete (... optimized for each transfer plasmid. Pro-Tip Endotoxins can inhibit transfection; therefore, plasmid ...

...ensure that you are in compliance with your institution’s biosafety regulations. Last Update: February... °C. See below for other Reagent Preparation instructions. Wipe down all pipettes and equipment with 10%...

...purification and isolation of cells, organelles, lipoproteins, and macromolecules. Importantly, iodixanol ...of contaminants in each fraction. * AAV capsid proteins VP1, VP2, VP3; M protein marker. Image adapted...

...cloning cylinders. Workflow Timeline This protocol begins with a stable cell pool. For details, see our protocol...homogenized cell solution to 990 µL complete medium. Instead of transferring 0.125 µL, now transfer 100 times...

...you have a NanoDrop, follow the manufacturer's instructions for the spectrophotometer specific to your lab...

...Extraction Kit . Always follow the manufacturer's instructions. Note: It is usually important to determine ...

...ensure that you are in compliance with your institution’s biosafety regulations. Workflow Timeline Day...

...µg of DNA into 500 µL of Opti-Pro SFM. Pro-Tip Endotoxins can inhibit transfection; therefore, plasmid ...

...the liquid and avoid touching the pipette to the inside of the tube. Only the tip should be in the liquid...

...into polypeptide chains that make up the primary structure of proteins. Most proteins are then modified...systems. Insect expression systems Insect cells can also be used to produce complex eukaryotic proteins with...Researches can visualize proteins in vivo by tagging them with fluorescent proteins to study localization...localization or purify proteins to study their structure, interactions and functions. Proteins can also be purified... to manipulate DNA), or in medicine (e.g. insulin). Proteins, unlike DNA which can be relatively easily...are two types of insect expression systems; baculovirus infected and non-lytic insect cells. Baculovirus...genes integrated into the insect cell genome. Both of these types of insect expression systems can be ...

...       Insert-specific primers: Insert-specific primers are designed to anneal to an insert-specific... your insert or if the insert is in your plasmid Have to make a new primer pair for each insert Backbone-Specific...the insert, insert orientation, and the sequences of the junctions between the plasmid and insert DNA....negative clone without the insert. This type of primer pair can tell you if the insert is the correct size and...orientation of the insert. Orientation-specific primers: If you need information about insert orientation,...of the insert. One member of this type of primer pair anneals to a sequence flanking the insert and one...anneals to the insert. A simple way to create this type of primer pair is to mix-and-match insert-specific ...

...reporters as well as secretory proteins or proteins targeted to membrane sub-domains. It has also been used to... reporter mRNA, which contains the tethering sites for the fluorescent proteins and an inducible promoter...and the RFP proteins in the nucleus. During the first round of translation, the GFP proteins that are bound...the nucleus (thanks to a NLS on these proteins). The RFP proteins, however, remain bound to the mRNA since...the Wyss Institute have extended the transactivating capabilities of CRISPR/dCas9-fusion proteins and have...engineered hook proteins fused to a streptavidin core that are able to retain reporter proteins fused to the...possible protein domains containing these properties, they generated monoclonal antibodies against them, fused...

... open a plasmid (backbone) and insert a linear fragment of DNA (insert) that has been cut by compatible... allow your insert to be placed into the backbone in the proper orientation. For instance, if you were...enzyme if your insert is flanked on both sides by that enzyme’s restriction sites, but the insert can then ...used. Follow the manufacturer’s instructions.   2. Isolate Your Insert and Vector by Gel Purification ...separate your backbone away from any inserts cut out of it and your new insert from any overhangs cut off of ...these stains see the Bitesize Bio Blog and their associated manufacturers websites. These stains require...Your Insert into Your Vector In the ligation step, you mix your purified, cut backbone and insert in a...

...fused to two proteins, here proteins Y and Z, that are known to interact. When  proteins Y and Z interact...interacting proteins; nearby proteins are biotinylated when and where the two bait proteins interact, but...interact or from proteins that are just in the proximity of the bait proteins. Instead of pinpointing specific...restored, and nearby proteins are labeled with biotin. For labeling, BirA* fusion proteins are expressed in...activity is restored, and nearby proteins are labeled with biotin. When proteins Y and Z part ways, BirA* biotinylation...the biotin labeling remains even if the interaction with the BirA* fused bait proteins ceases. Lastly, Split-BioID...provides a candidate list of proteins that are part of the bait proteins’ interactome. Further characterization...

.... The Moffat laboratory maintains a website for the TKO library that contains up-to-date information and...(3) but its portability to other bacterial strains remains seriously limited, as prior optimizations are...ligase to biotinylate proximate proteins in the cell. These candidate proteins can be enriched via biotin ...of fusion proteins, requires less biotin, and shows enhanced labeling of proximate proteins. Overall BioID2...optimization of industrial yeast strains. However, industrial yeast strains are more difficult to genetically...which “bait” proteins are tagged with Protein A-Renilla luciferase and the “prey” proteins are tagged with...of RNA-binding proteins identified by editing Identifying targets of RNA-binding proteins (RBPs) has traditionally...

... next insert. Repeat as needed for more inserts. When you are finished entering your gene/insert data,...cloning a gene/insert into it. Empty backbones often contain epitope tags or fusion proteins that are appended...within your insert (or targeted by a gRNA/shRNA) and select the species that the gene or insert sequence ...include the total size of the insert and the GenBank ID associated with your insert. Pro Tip! If the correct...of the insert (when cloned into the vector backbone). If your plasmid has more than one insert, click .... For instance, if your plasmid is designed to allow you to produce large amounts of your insert in E....it. If your plasmid has multiple inserts, enter details for each insert individually.   Figure 9: Add ...

... either side of the expected insert. If the plasmid contains your insert, you will see two bands: one ...another enzyme that cuts within the insert. If the plasmid contains the insert, you will see two bands but if...the plasmid contains the insert and no PCR product if the plasmid does not contain the insert. If you’re...but it won’t tell you whether the vector contains the gRNA insert or not. A blue-white screen, however, ...sized bands depending on whether the plasmid contains your insert of interest or not. For example, choose ...the other representing the insert. If the plasmid doesn’t contain the insert, you will just see the vector...PCR to detect your insert, either by using primers that are specific to your insert or by using primers...