Addgene is a nonprofit plasmid repository. We store and distribute high-quality plasmids from your colleagues Plasmid pUC19_CRISPR_DpmrA from Dr. Anne-Catrin Uhlemann's lab contains the inserts Cas9, Lambda Red Recombineering Genes, and Zeocin Resistance Cassette and is published in Cell Rep. 2020 Oct 27;33(4):108313. doi: 10.1016/j.celrep.2020.108313. This plasmid is available through Addgene Plasmid pUC19 - T7 pro - IRES - EGFP from Dr. Fei Chen's lab contains the insert EGFP and is published in Nat Biotechnol. 2019 Dec 16. pii: 10.1038/s41587-019-0331-8. doi: 10.1038/s41587-019-0331-8. This plasmid is available through Addgene
Plasmid pUC19-gRNA from Dr. Kabin Xie's lab contains the insert gRNA scaffold and is published in Unpublished This plasmid is available through Addgene. Image: Illustrated plasmid map in PNG format GenBank File: Plasmid sequence and annotations
Plasmid pUC19-PpSEC13-mEGFP from Dr. Benjamin Glick's lab contains the insert ppSEC13-mEGFP and is published in Traffic. 2020 May 15. doi: 10.1111/tra.12737. This plasmid is available through Addgene . Die Gene für die Ampicillinresistenz, den Replikationsursprung sowie der Polylinker (blau) sind eingezeichnet. pUC19 gehört wie auch pUC18 zu einer Reihe im Labor von Joachim Messing gentechnologisch hergestellter Klonierungsvektoren
pUC19 is a commonly used cloning vector that conveys the Amp resistance. The molecule is a small double-stranded circle, 2686 base pairs in length, and has a high copy number. pUC19 carries a 54 base-pair multiple cloning site polylinker that contains unique sites for 13 different hexanucleotide-specific restriction endonucleases (1) pUC19. Standard E. coli vector with a multiple cloning site (MCS) for DNA cloning. The MCS is reversed in pUC18. To see this sequence with restriction sites, features, and translations, please download SnapGene or the free SnapGene Viewer. pUC19.dna *Note: Two Popular Vectors for Blue White Screening Available at Addgene Are: pUC18 and pUC19 Let's begin at the beginning. The well-characterized bacterial lac operon contains a gene called lacZ that encodes for the enzyme β-galactosidase
Title: pUC19-gRNA.tif Created Date: 12/9/2019 3:27:58 P pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers. The designation pUC is derived from the classical p prefix (denoting plasmid ) and the abbreviation for the University of California, where early work on the plasmid series had been conducted
Import Plasmid mUAV (addgene) and Plasmid pUC19 (addgene) into Benchling. Benchling tutorials; Restriction digest pUC19 with PvuII and identify the backbone you want to use for your assembly. [Hint: You need a selection marker and origin of replication!] Identify the gene encoding for chromophore, RBS, promoter, and terminator in Plasmid mUAV. In mUAV plasmid, if you change bases from 2309. DNASU and Addgene are central repositories for plasmid clones and collections that may also be helpful. Product Source pUC19 is isolated from E. coli ER2272 (dam dcm EcoK M-) by a standard plasmid purification procedure. Polylinker DNA Sequenc pUC19 Homology to AAVS1 Homology to AAVS1 SA SV40 PolyA BGH PolyA MCS MCS . Created Date: 7/28/2015 3:54:41 PM. home > Addgene > puC19 preACT1(BrC) product summary. company name : Addgene. product type : cDNA. product name : puC19 preACT1(BrC) catalog : 111325. more info or order : Addgene product webpage . citations: 1. Reference; Villa T, Guthrie C. The Isy1p component of the NineTeen complex interacts with the ATPase Prp16p to regulate the fidelity of pre-mRNA splicing. Genes Dev. 2005;19:1894-904.
Addgene is a nonprofit plasmid repository. We archive and distribute high quality plasmids from your colleagues pUC19 Standard E. coli vector with a multiple cloning site (MCS) for DNA cloning
Verify the sequence of each colony by sequencing from the pUC19 backbone using the pUC19-Fwd or pUC19-Rev primer. Reference the sequencing results against the expected genomic sequence to check for the presence of Cas9-induced NHEJ or HDR modifications. Calculate the percentage of editing efficiency as (no. of modified clones)/ (no. of total clones). It is important to pick a reasonable number. For control: Add 1 µL (10 ng) pUC19 control DNA to one tube. Mix by gentle tapping and place on ice: Add 1 ng to 50 ng of purified plasmid DNA directly to cells in rest of the tubes. Mix by gentle tapping and place on ice: Incubate the cells on ice for 30 minutes: Transfer the cells to 37° C water bath for exactly 45 seconds: Transfer the cells to ice for 2 minutes: Add SOC medium to each. . CRISPR-Cas9 (Cas9) is an RNA-guided nuclease that targets and cuts genomic DNA. The interplay between Cas9 (which causes the breaks) and host cell DNA repair factors (which repair those breaks) makes Cas9 extremely effective as a genome editing reagent pUC19(ColE1*ori) (the*RK2*ori*taken*from* pBIN19*vector*is*not necessary*in*this* backbone,*butjust happens*to*be*there) ' TTAC ACTA GCAA CAGA TGTG GAGC TGCC Other*vectors* pICH82094 KanR* *LB* *RB* DraIIIDraIII Fori* RiA4 ori* TGCC*GGGA I * ColE1 I * pICH89921 Z* p15A* ori* KanR* *LB* *RB* DraIII DraIII TGCC* GGGA I * aI * aI * ColE1 Z* Rk2*oriV* Rk2*trfa* pICH81091* pICH81071.
You've worked hard to purify your gene of interest, get it into your plasmid backbone, and zap the mixture of DNA into cells.Unfortunately, not every cell successfully takes up plasmid DNA. Among those that do, some now have plasmids that contain your gene of interest, but others will uptake plasmid backbones that re-ligated back on themselves Here at Addgene, we use NGS-based quality control to confirm the sequence of all the plasmids we distribute. This method is time-intensive, so we recommend a variety of ways to screen and verify your plasmids. Here, we'll cover restriction digest analysis. Diagnostic restriction digest . Diagnostic digests can be used to confirm the rough structure of the plasmid based on the predicted sizes. A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. The purpose of a MCS in a plasmid is to allow a piece of DNA to be inserted into that region
Fullconstruct Pax6_pUC19 2 - 7158 nt . Title: FullConstruct_Pax6_pUC19_2-1 Author: Pepe-Caprio, Angela L./Sloan-Kettering Institute Created Date: 12/7/2017 8:08:21 PM. Note: For control: Add 1µL (10ng) pUC19 control DNA to one tube. Mix by gentle tapping and place on ice. Incubate the cells on ice for 30 minutes. Transfer the cells to 37°C water bath for exactly 45 seconds. Transfer the cells to ice for 2 minutes. Add SOC medium to each tube. Transfer the cells to sterile polypropylene tubes and loosen the caps to facilitate aeration of the cultures. SaCas9 was cloned from pX601 plasmid (a gift from Feng Zhang, Addgene plasmid #61591) into a vector with a strong CAG promoter to enable robust expression in hiPSC (CAG-SaCas9, deposited in Addgene). SaCas9 guide RNAs targeting SOX2 stop codon were designed using Benchling. Guide-RNA transcriptional cassettes were assembled by PCR as previously described Balboa et al., 2015). pUC19-SOX2-T2A. CRISPR plasmids for genome editing and gene regulation from Addgene, transOMIC, and others. Fluorescent Protein Genes & Plasmids. Commonly used fluorescent protein genes and vectors. Gateway® Cloning Vectors. Commonly used Gateway® sequences including Donor Vectors, Entry Vectors, and Destination Vectors. I.M.A.G.E. Consortium Plasmid Using 10 ng-1000 ng of clean, supercoiled pUC19 or pUC19 isolated with a commercial miniprep kit, total colonies increase with increasing DNA concentration. * Ideally, DNA for transformation should be purified and resuspended in water or TE. However, up to 10 µl of DNA directly from a ligation mix can be used with only a two-fold loss of transformation efficiency. Where it is necessary to.
The GFP gene was inserted in frame with the lacZ initiation codon from pUC19 so that in E. coli, GFP is expressed from the lac promoter as a fusion with several additional amino acids, including the the first five amino acids of the lacZ protein. Note, however, that if you excise the GFP coding sequence using a restriction site in the 5' MCS, the resulting fragment will encode the native (i.e. .. MDA assays and fidelity analyses were carried out as described DNASU and Addgene are central repositories for plasmid clones and collections that may also be helpful. Product Source pBR322 is isolated from E. coli ER2686 (dam +dcm + EcoK M-) by a standard plasmid purification procedure. Reagents Supplied . The following reagents are supplied with this product: Show all Collapse all. NEB # Component Name Component # Stored at (°C) Amount Concentration. A) NEB Stable competent cells or B) Stbl3 competent cells were transformed with 2 µl of a pUC-5xREP Gibson Assembly reaction containing 2.2 ng (0.00125 pmol) pUC19 vector and approximately 1 ng (0.0028 pmol) 5xREP insert. Transformed cultures were plated on LB plates containing 100 µg/ml ampicillin and incubated overnight at 30°C. The next day, colony PCR was performed using M13/pUC.
Translational reporters are in-frame gene fusions between GFP and a gene of interest (Figure 1B). Ideally, a translational reporter includes the entire genomic locus of a gene (5 ′ upstream region, exons, introns, 3 ′ UTR). GFP can be inserted at any point in the open reading frame, preferably at a site that does not disrupt protein function or topology Addgene
. Turn on electroporator and set to 1.7-2.5 kv (optimize for strain), 200 ohms and 25 µF. Place recovery SOC in 37°C water bath. Pre-warm LB-antibiotic plates at 37°C. Thaw cells on ice for 10 min or use freshly made cells. Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice. Flick. Addgene Cat# 164441: pHDM-Tat1b (helper) This study: Addgene Cat# 164442: pRC-CMV-Rev1b (helper) This study: Addgene Cat# 164443: pUC19 (empty) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Norrander J, Kempe T, Messing J. Gene. 1983 Dec;26(1):101-6. 10.1016/0378-1119(83)90040-9: Joachim Messin pUC19 Control DNA . 10 pg/μL in 5 mM Tris-HCl, 0.5 mM EDTA, pH 8 . 50 μL . S.O.C. Medium . 2% Tryptone 0.5% Yeast extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl. 2 10 mM MgSO 4 20 mM glucose . 6 mL . Genotype of TOP10 Strain . F- mcrA ∆(mrr-hsdRMS-mcrBC) Φ80lacZ∆M15 ∆lacχ74 recA1 araD139 ∆(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG . Product Use . For Research Use Only. Not for use in.
חוליה מקשרת (MCS, Multiple Cloning Site), המכונה גם פולינלינקר ואתר בעל אתרי שיבוט מרובים, היא שמו של מקטע קצר של DNA מסונתז בפלסמידים מהונדסים המכיל אתרי קיטוע רבים של אנזימי הגבלה שונים (עד 20 אתרים). החוליה המקשרת מסייעת בהחדרת. Protocol:propagationofgenome1widelentiviralCRISPR1guide(RNAlibraries((Version201411114(! KosukeYusa(email@example.com)! Wellcome!Trust!Sanger!Institute .025 ml 50 pg/µl SOC Outgrowth Medium: B9020SVIAL: 4 1 x 25 ml Not Applicable : Gibson Assembly Master Mix: M5510AVIAL-20 1 x 0.1 ml 2 X Product Categories: DNA Assembly, Cloning and Mutagenesis Kits Products Applications: Gibson Assembly® Advantages and Features. Features. Assembly and transformation in just under two hours; Flexible sequence design (scar.
3WJdB 294..448 Ampicillin Resistance 1113..1973 pBR322 origin 2128..2747 pUC19-T7-3WJdB-T 2914 bp XbaI restriction site 257..262 T7 Promoter 277..29 Completed Constructs. We have transformed Aurantiochytrium using an expression construct for zeocin resistance (shble) driven by Aurantiochytrium GAPDH promoter and terminator sequences - the GZG cassette - cloned into pUC19, with and without a copy of the Aurantiochytrium 18S rRNA gene. These two plasmids, pUC19_GZG and pUC19_18GZG respectively, are available at addgene Bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site. For other reading frames, use pET-28b(+) or pET-28c(+) ベク pUC19 Vector pUC19 Vector pUC19 Vector カタログ番号: N3041 カタログ番号 サイズ 濃度 価格 保存温度 N3041S 50 μg 1,000 μg/ml ¥16,000-20C N3041L 250 μg 1,000 μg/ml ¥64,000-20C 製品カテゴリ>グループ DNA修飾酵素 特徴. 実験に使う試薬 (a) 10x 制限酵素バッファー 10x というのは，反応液中の最終濃度に対して 10 倍の.
You then insert this gene into the Pst1 site on pUC19 and transform the recombinant plasmid into E. coli. A. Outline (using a well-labeled sketch) the general transformation and selection protocol. B The fragments were then ligated using T4 ligase (NEB) and cloned into the BsaI-digested pUC19-based hU6-pegRNA-gg-acceptor entry vector (Addgene no. 132777). For nicking gRNA (ngRNA) cloning. pMAL-c5X Bacterial vector for inducible cytoplasmic expression of maltose-binding protein (MBP) fusions with a Factor Xa cleavage site
The map, notes, and annotations on this page and in the sequence/map file are copyrighted material ③Addgene下载的pUC19序列（标准序列）； 方法（详细操作步骤）： ①点击上述网址，勾选Align two or more sequences： ②在Enter Query Sequence中输入金唯智M13F(-47)正向引物测序结果（正反向均可，NCBI比较智能，可以自行调整）： ③在Enter Subject Sequence中输入Addgene下载的pUC19序列： ④ 继续下滑.
pJET1.2/blunt Linearized positive selection cloning vector with a lethal insert that allows for efficient recovery of blunt-ended PCR products One Shot® TOP10F´ Chemically Competent E. coli are identical to TOP10 cells, with the addition of an F´ episome. TOP10 competent cells are provided at a transformation efficiency of 1 x 10 9 cfu/µg supercoiled DNA and are ideal for high-efficiency cloning and plasmid propagation. Using One Shot® TOP10F´ Chemically Competent E. coli The F´ episome carries the tetracycline resistance gene. The three lentivirus construction plasmids are all available from Addgene; the HyPer2 plasmid is from Evrogen. The pWPXL plasmid vector comes with an eGFP insert; methods for exchanging the eGFP insert for the gene of interest can be found in standard molecular biology protocol reference. The vector comes in two easy to confound variations (pWPXL and pWPXLd); the difference is in the loxP. ST19 + pUC19 S. typhimurium ST19 Δ mfd::cat + pUC19 Transformation of pHM651 into HM3429 HM2212 HM2260 MR01 Sherman lab stock MR02 Recombineering into MR01 using pNIT. Electroporation of hygromycin marked amplicon using primers HM2877 and HM3908 and pHM566 as template HM2747 mini λ Red recominbase expression plasmid for recomineering in S.
pUC19 E. coli cloning vector (pMB1 ori; Apr; lacZ=) Laboratory stock and reference 43 pKD46 E. coli lambda Red recombineering expression vector (pSC101ts; Apr; araC-P araB-gam-bet-exo) E coli Genetic Stock Center and reference 3 pCas9 E. coli vector containing the S. pyogenes cas9 gene and tracrRNA (p15A ori; Cmr; cas9; tracrRNA) Addgene and. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell). pSB1C3 has terminators bracketing its MCS which are designed to prevent transcription from *inside* the MCS from reading out into the vector. The efficiency of these terminators is known to be < 100%. Ideally we would construct a future set of terminators for bracketing a MCS that were 100% efficient in. We recommend including the pUC19 control plasmid DNA supplied with the kit (10 pg/μL in 5 mM Tris-HCl, 0.5 mM EDTA, pH 8) in your transformation experiment to verify the efficiency of the competent cells. Do not use these cells for electroporation. 1. Thaw, on ice, one vial of One Shot™ Stbl3™ chemically competent cells for each transformation. 2. Add 1 to 5 μL of the DNA (10 pg to 100. Plasmids used to target ROSA26 (pEN111) and TIGRE (also known as Igs7) (Addgene 92141) loci, and the ROSA26- and TIGRE-specific sgRNA-encoding plasmids (Addgene 86234 and 92144, respectively) were.
Transformation efficiency: 1-3 x 10 6 cfu/μg pUC19 DNA; Activity of nonspecific endonuclease I (endA1) eliminated for highest quality plasmid preparations; K12 Strain; Genotype ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm-6 hisG4 rfbD1 R(zgb210::Tn10) Tet S endA1 rspL136 (Str R) dam13::Tn9 (Cam R) xylA-5 mtl-1 thi-1 mcrB1 hsdR2 Reagents Supplied . The following reagents are. Advantages of Custom Gene Synthesis. Any DNA sequence, even complex, GC-rich, repeated, or long genes, with 100% sequence accuracy guaranteed. Delivery in as few as 4 days -- the fastest turnaround time in the industry. First response team and local facility allows fastest delivery; Data security meets information regulatory Title: AKT/protein kinase B associates with β-actin in the nucleus of melanoma cells. Report recurrent GLI1-ACTB gene fusions in a group of malignant mesenchymal neoplasms involving soft tissue, and occasionally bone, with an often nested epithelioid phenotype and strong S100 immunoreactivity One Shot Stbl3 Chemically Competent E. coli are designed especially for cloning direct repeats found in lentiviral expression vectors. Unlike TOP10 E. coli, these cells reduce the frequency of homologous recombination of long terminal repeats (LTRs) found in ViraPower Lentiviral Expression Vectors Use NEBcloner to find the right products and protocols for each in your traditional cloning workflow, including double digestion buffers
ACTB (Actin Beta) is a Protein Coding gene. Diseases associated with ACTB include Dystonia, Juvenile-Onset and Baraitser-Winter Syndrome 1.Among its related pathways are G-Beta Gamma Signaling and Apoptosis Modulation and Signaling.Gene Ontology (GO) annotations related to this gene include identical protein binding and RNA polymerase II proximal promoter sequence-specific DNA binding As a leading biotech company focusing exclusively on early drug discovery and development services, GenScript provides a comprehensive portfolio of services that include Bio-Reagent, Bio-Assay, Lead Optimization, and Antibody Drug Development
Wild type SAE2 and UBC9 ORF were amplified by RT-PCR using Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific) and subcloned into pUC19 vector using InFusion HD Cloning Kit (Takara Bio USA). Both, SAE2 and UBC9 ORFs were then PCR amplified and subcloned into pLV-mCherry plasmid (Addgene #36084). All PCR steps described above have been performed using Phusion Hot Start II DNA. Assembly of these elements into the pUC19 derivative plasmid pNIM14 was previously described (Naduthodi et al., 2019). Using pNIM14 as a template, we constructed pNIM21 by adding terminal MmeI restriction endonuclease recognition sequences to facilitate precise excision of the IC from genomic DNA during insertion site tracing Conventional CRISPR-Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we.
ASPM (known as Asp in fly and ASPM-1 in worm) is a microcephaly-associated protein family that regulates spindle architecture, but the underlying mechanism is poorly understood. Here, we show that. Benoit et al. screened extracts from strains of actinomycete to uncover and isolate a molecule that inhibits SUMOylation in human cancer stem cells. This natural product compound acts via biochemical targeting of a specific motif in a SUMOylation enzyme, SAE2, that regulates self-renewal of a variety of human cancer cells