... easily gate on the unlabeled cells of interest while the rest can be excluded, or “dumped”. From left...fluorophore-conjugated antibodies directly bind to epitopes of interest that are found in the extracellular space. The...like bulk lympho-/splenocytes, you might only be interested in one of the various cell populations; for example...selection: labeling markers that your cells of interest do not have, and selecting for the cells that ...quick method of negatively selecting your cells of interest is magnetic bead-activated cell sorting, or MACS...An effective strategy to sort out your cells of interest via FACS using negative selection is called dump...Intracellular labeling Oftentimes, a marker of interest is not located on the cell membrane but inside...

...between cancer cells, therapeutic drugs, and the rest of the body than studies in other model organisms...level and spatiotemporal expression of a gene of interest. This type of control over gene expression is ...continuously improved upon since then. If you are interested in these systems check out Gerald Rubin’s lab...specific plasmids (L4440) that contain your gene of interest (Fire et al., 1998). The discovery of RNAi in ...sensor, GCaMP6 (Nguyen et al., 2015). If you are interested in C. elegans plasmids and resources check out...

...-CDS issues. Prohibited Restriction Sites: EcoRI, XbaI, SpeI, PstI Restriction Sites to Avoid: NotI For...Coli genome. Prohibited Restriction Sites: EcoRI, SpeI, NheI, PstI Restriction Sites to Avoid: PvuII, ... use of assembly standards – combinations of restriction sites which preserve a standard prefix and suffix...backbone can be repeated to ligate your gene of interest after the promoter, and so on. The following table... reverse complimented for primer design. The restriction enzymes used for assembly are the prohibited ... Employs high-efficiency enzymes. Prohibited Restriction Sites: EcoRI, BglII, BamHI, XhoI For more info...giving in-frame scar encoding Thr-Ala. Prohibited Restriction Sites: EcoRI, XbaI, SpeI, PstI For more info,...

...Experimental Tips Choosing a tet system If your gene of interest should be active, and only turned off occasionally...off or tTA is more appropriate. If your gene of interest should be mostly inactive, and only turned on ...tetracycline-dependent promoter upstream of your gene of interest tTA expression plasmid Components for a tetracycline...tetracycline-dependent promoter upstream of your gene of interest rtTA or TetR expression plasmid Stable cell lines...will allow you to insert and induce your gene of interest. ID Plasmid Description Co-expressed tTA, rtTA... 16542 pBI-MCS-EGFP Expression of your gene of interest (MCS with a β-globin poly A) & EGFP from a bidirectional...plasmid for inducible expression of transgene of interest and EGFP None Either Lung 11662 pPRIME-TET-GFP-FF3...

...nucleotide edits that introduce a novel restriction site. The region of interest is PCR amplified, digested with...region of interest can be PCR amplified using primers that (A) flank the region of interest (deletions...necessary to model your specific disease or process of interest. Do you want to: Generate complete and permanent...the promoter driving expression of your gene of interest. For genetic knockouts, gRNAs commonly target ... containing the gRNA scaffold using standard restriction-ligation cloning. However, the exact cloning ... within a mixed cell population. The region of interest is PCR amplified, PCR products are denatured-renatured...agarose gel to identify DNA fragments. PCR and restriction digest (for HDR repaired DSBs): For small nucleotide...

...analysis to purify specific genomic regions of interest 48807 8xLexA-binding elements/pMD20 iChIP plasmid...analysis to purify specific genomic regions of interest 51258 3xFLAG-dCas9/pMXs-IG Expresses 3xFLAG-dCas9...analysis to purify specific genomic regions of interest 51259 3xFLAG-dCas9/pMXs-I2 Expresses 3xFLAG-dCas9...analysis to purify specific genomic regions of interest 51260 3xFLAG-dCas9/pMXs-neo Expresses 3xFLAG-dCas9...analysis to purify specific genomic regions of interest 61079 gRNA-hIRF-1 #12 Expresses a guide RNA (gRNA...analysis to purify specific genomic regions of interest 63589 3xFN-Tel-TAL/pCMV-7.1 Expresses a 3xFLAG-tagged...analysis to purify specific genomic regions of interest 82613 3xFLAG-dCas9/MSCV-EGFP Expresses 3xFLAG-...

...can precisely control expression of your gene of interest. Many Cre constructs also contain fluorescent ...a promoterless vector with several available restriction sites none Mammalian Sauer 11957 pBS595 tet-hCMV-EGFPcre...with stop codon Entry vector Ko 139546 pENTR-CreStopDeleted Cre without stop codon Entry vector Ko 139547...-lox” or “LSL” cassette) upstream of a gene of interest will prevent gene expression in the absence of... Switch: These constructs contain two genes of interest, Genes A and B. When Cre is absent, only Gene ...and type of target sites that flox your genes of interest makes FLEx switch a powerful experimental tool...information. Empty backbones to regulate your gene of interest: ID Plasmid Description Expression System PI 11578...

...Express gene of interest from the CMV promoter pCDH-EF1 72266 Express gene of interest from the EF-1 promoter... Express gene of interest from the CB promoter pCDH-PGK 72268 xpress gene of interest from the PGK (phosphoglycerate...Express gene of interest from the CB promoter pCDH-EF1s 72484 Express gene of interest from a truncated...respectively, and empty vectors to express your gene of interest under the control of a variety of promoters. Professor...kinase I) promoter pCDH-CMV4 72284 Express gene of interest from the CMV promoter. WPRE has been removed to...

...well as plasmids that already contain a region of interest before or after luciferase . In addition, Addgene... pGL3-LRR : Genomic tiles spanning a region of interest are cloned into a Firefly luciferase reporter ...you can clone your regulatory element or gene of interest into to create a luciferase reporter. Unless otherwise...Lentiviral expression of firefly luciferase and gene of interest Bryan Welm 18760 MSCV IRES Luciferase Firefly ...Retroviral expression of firefly luciferase and gene of interest Scott Lowe 18782 MSCV Luciferase PGK-hygro Firefly...expression of firefly luciferase and dTomato. A gene of interest can also be inserted into the plasmid. Malcolm...construct that contains your regulatory element of interest. ID Plasmid Description Gene/Insert PI Return ...

... the experiment, preventing contamination of the rest. Avoid long exposure of the cell culture to air ...

...out my window and I saw the Dorr Mountain with the rest of the skyline of Acadia National Park. I was confused...molecular biologist with Biogen and is particularly interested in applying CRISPR for mouse genome editing. ...Zoology, Chinese Academy of Sciences, and he is interested in using CRISPR to develop therapeutic applications...

...acids (ncAAs). A plasmid containing the gene of interest with the modified codon (typically the amber codon...a non-canonical amino acid into the protein of interest: The non-canonical amino acid, which is generally...codons, this can be challenging. In E.coli , the rarest codon is the amber stop codon (UAG) and thus this...this codon is often used. The gene of interest can be expressed from a plasmid containing a UAG codon ...pEVOL-NBK-1 PylRS M. mazei Pyrrolysine Bacterial Cole DeForest 209738 pAB228i16 G1 PylRS Methanogenic archaeon...sites changes to UAG, RF1 function removed, MutS restored so decreased mutation rate George Church 68306...

...Enzyme Collection DNA Ligases DNA Polymerases Restriction Endonucleases Reverse Transcriptases RNA Polymerases...polymerases, DNA ligases, reverse transcriptases, and restriction enzymes. These enzymes are useful in both basic...the links below: DNA Ligases DNA Polymerases Restriction Endonucleases Reverse Transcriptases RNA Polymerases...-phi29pol phi29 DNA Polymerase Return to top Restriction Endonucleases Addgene ID Plasmid Gene/insert ...

... incorporation phosphoserine into your gene of interest....choosing plasmids to express the phosphoprotein of interest, a medium copy RSF1030 origins or a low copy p15a...domain and the phosphosite, mCherry fluorescence is restored, so interactions can be discovered by fluorescence-activated...identify candidate human substrates of kinases of interest by mass spectrometry in a technique called serine-oriented...

... or by RT-PCR. Isolate your PCR product from the rest of the PCR reaction using a kit, such as the (Link...assist with restriction enzyme digestion (usually 3-6bp). Restriction Site: Your chosen restriction site for...fusing the upstream restriction site to the forward primer and the downstream restriction site to the reverse...product is now ready for restriction digestion. Digest Your DNA Set up restriction digests for your PCR product... Plasmid Cloning by PCR You may also like... Restriction Digest of Plasmid DNA DNA Ligation Bacterial ...of a piece of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that...that it can be easily cloned into a plasmid of interest. For this example, we will describe how to copy...

... The following resources may be of interest for groups interesting in implementing intersectional experimental...strategies to define cellular sub-populations of interest, including dual-transgenic recombinase-expressing...testing of novel INTSRECT plasmids for groups interested in producing their own INTRSECT plasmids. Online...

...step 3.1.4 ), 5.0 μl restriction enzyme buffer (10x), 4.0 μl (20 U) BbsI restriction enzyme (5,000 U/ml)...for cloning into the pX330 vector using BbsI restriction enzyme ( Table 3 ). Add a G nucleotide after ... construct. Transfecting CRISPRs into Cells of Interest NOTE: This protocol involves the delivery of CRISPR...

...Plasmids for REAL (Restriction Enzyme And Ligation) TALEN assembly containing single TALE repeat domains...any desired sequence, referred to as the REAL (Restriction Enzyme And Ligation) assembly method ( Sander...encoding a single TALE repeat domain and flanked by restriction sites used for ligation into longer arrays. These...

...various fluorescent markers and tag your proteins of interest with fluorescent proteins in yeast.... overexpression of FPs and tagging of genes of interest with FPs in yeast. Characterization of some of...tagging vectors to create fusions of proteins of interest with yeast codon-optimized fluorescent proteins...

...it's important to determine where your protein of interest resides. There are a variety of methods for determining...to compare the localization of your protein of interest with known markers. Addgene has assembled a collection...subcellular structures. You should fuse your protein of interest with a fluorescent protein that has a different...localization or possible function of a protein of interest. Browse or search the table below to find markers...