DDK phosphorylation of Mcm2 is required for cell growth. Protein expression and purification- Mcm2: Full-length Mcm2 and fragments were

Size: px
Start display at page:

Download "DDK phosphorylation of Mcm2 is required for cell growth. Protein expression and purification- Mcm2: Full-length Mcm2 and fragments were"

Transcription

1 DDK phosphorylation of Mcm2 is required for cell growth Supplemental Data Protein expression and purification- Mcm2: Full-length Mcm2 and fragments were cloned into pet33b (NdeI/BamHI). The plasmids were transformed into Escherichia coli Rossetta TM 2(BL21) (EMD Biosciences) and a colony from transformation was used to inoculate 6 L of ZYM-5052 auto-inducing media (Studier, 2005) containing 100 µg/ml kanamycin and 34 µg/ml chloramphenicol. The cells were grown at 37 C until an A 600 of 0.6 was reached, the temperature was then lowered to 14 C and the cells were allowed to express protein for 16 hours. Harvested cells were lysed by French press in 200 ml of 50 mm Tris-HCl ph 8.0, 500 mm NaCl, and 50 mm imidazole. The lysate was then applied to a 20 ml NiSO 4 -charged chelating Sepharose fast flow resin (GE Healthcare). The column was washed with the same buffer. The protein was then eluted in buffer 50 mm Tris-HCl ph 8.0, 100 mm NaCl, 10% glycerol, and 250 mm imidazole. Peak fractions were then dialyzed against 50 mm Tris-HCl ph 8.0, 50 mm NaCl, 10% glycerol and applied to an 8 ml SourceQ resin (GE Healthcare). The protein was then eluted with a 20-ml linear gradient from 50 mm-500 mm NaCl. Peak fractions were pooled and frozen. GST-Dbf4-Cdc7 The S. serevisiae Dbf4 gene was cloned into the NcoI/BamHI sites of pet28a (Figure 1) or pet41a vector (Figures 2-6). The Cdc7 gene was cloned into the NdeI/BamHI sites of pet16b (Figure 1) or pet11a (Figure 2-6). The expression and purification of Cdc7 alone or Dbf4 alone were essentially the same as that described

2 above for Mcm2. For the co-expression of DDK (Dbf4-Cdc7, Figures 2-6), both plasmids were transformed into Escherichia coli Rossetta TM 2(BL21) and the transformation mixture was plated on ampicillin and kanamycin. A colony from transformation was used to inoculate 6 L of ZYM-5052 auto-inducing media (Studier, 2005) containing 100 µg/ml kanamycin, 200 µg/ml ampicillin, and 34 µg/ml chloramphenicol. The DDK protein complex was then purified essentially as described above for Mcm2. Mcm2-7 reconstitution and Radiolabeling- The Mcm2-7 complex was reconstituted as previously described (Davey et al., 2003), except the Mcm3 protein contained an N- terminal site for protein kinase A phosphorylation. The Mcm2-7 complex was radiolabeled with protein kinase A in a reaction volume of 100 µl that contained 2.2 µm Mcm2-7 protein in Kinase reaction buffer (5 mm Tris-HCl ph 8.5, 10 mm MgCl 2, 1 mm

3 DTT, 500 µm ATP, 100 µci 32 P-γ-ATP) containing 5 µg PKA. Reactions were incubated at 30 C for 1 hr. Plasmids used in this study: pib201 (prs416 CEN6/ARSH4 MCM2 URA3) pib202 (prs415 CEN6/ARSH4 GAL1::MCM2 LEU2) pib203 (prs415 CEN6/ARSH4 GAL1::mcm2 S164A LEU2) pib204 (prs415 CEN6/ARSH4 GAL1::mcm2 S170A LEU2) pib205 (prs415 CEN6/ARSH4 GAL1::mcm2 S164A,S170A LEU2) Strains used in this study: Strain Genotype Source of reference BY4743 MATa/α his 3 1 leu2 0 met15 0 lys2 0 Open Biosystems ura3 0 MCM2+/mcm2::KanMX4 RSY311 MATa bar1 trp1 leu2 ura3 can1 his6 cyh2 Hoang et al. (2007) RSY727 MATa bar1 trp1 leu2 ura3 can1 his6 cyh2 Hoang et al. (2007) mcm5-bob1-1 cdc7 ::HIS3 his3-1 IRB311 RSY311; mcm2 :: KanMX4(pRS416 This study CEN6/ARSH4 MCM2+Ura3) IRB727 RSY727; mcm2 :: KanMX4(pRS416 This study CEN6/ARSH4 MCM2+Ura3) Yeast strains bearing the bob1 mutation were generously provided by Robert Sclafani (Haase and Reed, 2002). Vectors and genomic DNA were acquired from ATCC. S. cerevisiae yeast strain S288C was used as a source of genomic DNA. PCR products were cloned into XhoI /BamHI restriction sites of prs415 and prs416 vectors. Mutations and deletions were verified by PCR and DNA sequencing.

4 RSY311 and RSY 727 strains were transformed with the plasmid pib201. Overlap PCR method was used to introduce mcm2::kanmx4 deletion into haploid strains RSY311 and RSY727. The template for PCR was genomic DNA from Strain BY4743 carrying the mcm2::kanmx4 deletion (MATa/α his 3 1 leu2 0 met15 0 lys2 0 ura3 0 MCM2+/mcm2::KanMX4, Open Biosystems). The following sets of primers were used to generate two PCR products; MCM2-150upsteam-forward (5 - ATA GGA CAA AAA ATT TCG AGA AAC GAA ACC AG-3 )/KanMX4-reverse(5 -CGA CAT TAT CGC GAG CCC ATT TAT ACC CAT-3 ) and MCM2-150downstream-reverse (5 - TTT AGT TGA ATT GAA ATT AAA GAT ACA GTG TAT AA-3 )/KanMX4-up (5 -ATG GGT ATA AAT GGG CTC GCG ATA ATG TCG-3 ). The full-length, gel purified PCR product was transformed into the yeast strains. Site-specific integration into MCM2 was confirmed by the inability of the strain to survive in the absence of wild-type MCM2 (pib201) and by PCR reactions using either set of primers mentioned previously. Expression of mcm2 mutants was confirmed by RT PCR. The mcm2 mutants were tested for function and survival in vivo using a plasmid shuffle assay. The mcm2 mutants were transformed into IRB 311(mcm2 ::KanMX4 + prs416/mcm2 + ), IRB 727(mcm2 ::KanMX4 mcm5-bob1-1 cdc7 ::HIS3 + prs416/mcm2 + ), and selected on SCM-Leu-Ura plates. Transformants were re-streaked on SCM-Leu-Ura (2% Dextrose) plates. Single colonies were grown in YPD overnight, and then 10 fold serial dilutions were plated on both SCM-Leu-Ura Gal (2% Galactose) plates and SCM-Leu-FOA Gal (2% Galactose) plates. Clones that carried mutant mcm2 and were FOA-resistant were used for phenotypic studies.

5 References Davey, M. J., Indiani, C., and O'Donnell, M. (2003). Reconstitution of the Mcm2-7p heterohexamer, subunit arrangement, and ATP site architecture. J. Biol. Chem. 278, Haase, S., and Reed, S. (2002). Improved flow cytometric analysis of the budding yeast cell cycle. Cell Cycle 1, Studier, F. W. (2005). Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41,

DNA Sample preparation and Submission Guidelines

DNA Sample preparation and Submission Guidelines DNA Sample preparation and Submission Guidelines Requirements: Please submit samples in 1.5ml microcentrifuge tubes. Fill all the required information in the Eurofins DNA sequencing order form and send

More information

10 µg lyophilized plasmid DNA (store lyophilized plasmid at 20 C)

10 µg lyophilized plasmid DNA (store lyophilized plasmid at 20 C) TECHNICAL DATA SHEET BIOLUMINESCENCE RESONANCE ENERGY TRANSFER RENILLA LUCIFERASE FUSION PROTEIN EXPRESSION VECTOR Product: prluc-c Vectors Catalog number: Description: Amount: The prluc-c vectors contain

More information

Inverse PCR & Cycle Sequencing of P Element Insertions for STS Generation

Inverse PCR & Cycle Sequencing of P Element Insertions for STS Generation BDGP Resources Inverse PCR & Cycle Sequencing of P Element Insertions for STS Generation For recovery of sequences flanking PZ, PlacW and PEP elements E. Jay Rehm Berkeley Drosophila Genome Project I.

More information

GENEWIZ, Inc. DNA Sequencing Service Details for USC Norris Comprehensive Cancer Center DNA Core

GENEWIZ, Inc. DNA Sequencing Service Details for USC Norris Comprehensive Cancer Center DNA Core DNA Sequencing Services Pre-Mixed o Provide template and primer, mixed into the same tube* Pre-Defined o Provide template and primer in separate tubes* Custom o Full-service for samples with unknown concentration

More information

PCR was carried out in a reaction volume of 20 µl using the ABI AmpliTaq GOLD kit (ABI,

PCR was carried out in a reaction volume of 20 µl using the ABI AmpliTaq GOLD kit (ABI, Supplemental Text/Tables PCR Amplification and Sequencing PCR was carried out in a reaction volume of 20 µl using the ABI AmpliTaq GOLD kit (ABI, Foster City, CA). Each PCR reaction contained 20 ng genomic

More information

pcas-guide System Validation in Genome Editing

pcas-guide System Validation in Genome Editing pcas-guide System Validation in Genome Editing Tagging HSP60 with HA tag genome editing The latest tool in genome editing CRISPR/Cas9 allows for specific genome disruption and replacement in a flexible

More information

pcmv6-neo Vector Application Guide Contents

pcmv6-neo Vector Application Guide Contents pcmv6-neo Vector Application Guide Contents Package Contents and Storage Conditions... 2 Product Description... 2 Introduction... 2 Production and Quality Assurance... 2 Methods... 3 Other required reagents...

More information

(http://genomes.urv.es/caical) TUTORIAL. (July 2006)

(http://genomes.urv.es/caical) TUTORIAL. (July 2006) (http://genomes.urv.es/caical) TUTORIAL (July 2006) CAIcal manual 2 Table of contents Introduction... 3 Required inputs... 5 SECTION A Calculation of parameters... 8 SECTION B CAI calculation for FASTA

More information

RT-PCR: Two-Step Protocol

RT-PCR: Two-Step Protocol RT-PCR: Two-Step Protocol We will provide both one-step and two-step protocols for RT-PCR. We recommend the twostep protocol for this class. In the one-step protocol, the components of RT and PCR are mixed

More information

UNIVERSITETET I OSLO Det matematisk-naturvitenskapelige fakultet

UNIVERSITETET I OSLO Det matematisk-naturvitenskapelige fakultet 1 UNIVERSITETET I OSLO Det matematisk-naturvitenskapelige fakultet Exam in: MBV4010 Arbeidsmetoder i molekylærbiologi og biokjemi I MBV4010 Methods in molecular biology and biochemistry I Day of exam:.

More information

Supplementary Online Material for Morris et al. sirna-induced transcriptional gene

Supplementary Online Material for Morris et al. sirna-induced transcriptional gene Supplementary Online Material for Morris et al. sirna-induced transcriptional gene silencing in human cells. Materials and Methods Lentiviral vector and sirnas. FIV vector pve-gfpwp was prepared as described

More information

Thermo Scientific Phusion Site-Directed Mutagenesis Kit #F-541

Thermo Scientific Phusion Site-Directed Mutagenesis Kit #F-541 PRODUCT INFORMATION Thermo Scientific Phusion Site-Directed Mutagenesis Kit #F-541 Lot _ Store at -20 C Expiry Date _ www.thermoscientific.com/onebio CERTIFICATE OF ANALYSIS The Phusion Site-Directed Mutagenesis

More information

Gene Synthesis 191. Mutagenesis 194. Gene Cloning 196. AccuGeneBlock Service 198. Gene Synthesis FAQs 201. User Protocol 204

Gene Synthesis 191. Mutagenesis 194. Gene Cloning 196. AccuGeneBlock Service 198. Gene Synthesis FAQs 201. User Protocol 204 Gene Synthesis 191 Mutagenesis 194 Gene Cloning 196 AccuGeneBlock Service 198 Gene Synthesis FAQs 201 User Protocol 204 Gene Synthesis Overview Gene synthesis is the most cost-effective way to enhance

More information

Supporting Information

Supporting Information Supporting Information Wiley-VCH 2007 69451 Weinheim, Germany Evolving a Thermostable DNA polymerase that Accurately Amplifies Highly-Damaged Templates Christian Gloeckner, Katharina B. M. Sauter, and

More information

TransformAid Bacterial Transformation Kit

TransformAid Bacterial Transformation Kit Home Contacts Order Catalog Support Search Alphabetical Index Numerical Index Restriction Endonucleases Modifying Enzymes PCR Kits Markers Nucleic Acids Nucleotides & Oligonucleotides Media Transfection

More information

TIANquick Mini Purification Kit

TIANquick Mini Purification Kit TIANquick Mini Purification Kit For purification of PCR products, 100 bp to 20 kb www.tiangen.com TIANquick Mini Purification Kit (Spin column) Cat no. DP203 Kit Contents Contents Buffer BL Buffer PB Buffer

More information

Updated: July 2005. 5' End label RNA markers 18.113 (18mer) and 44.12 (24mer) with Kinase and 32P-gamma-ATP. Gel purify labeled markers.

Updated: July 2005. 5' End label RNA markers 18.113 (18mer) and 44.12 (24mer) with Kinase and 32P-gamma-ATP. Gel purify labeled markers. RNA Cloning Method Flowchart Extract total RNA containing small RNAs. Check quality on denaturing gel with EtBr staining 5' End label RNA markers 18.113 (18mer) and 44.12 (24mer) with Kinase and 32P-gamma-ATP.

More information

TITRATION OF raav (VG) USING QUANTITATIVE REAL TIME PCR

TITRATION OF raav (VG) USING QUANTITATIVE REAL TIME PCR Page 1 of 5 Materials DNase digestion buffer [13 mm Tris-Cl, ph7,5 / 5 mm MgCl2 / 0,12 mm CaCl2] RSS plasmid ptr-uf11 SV40pA Forward primer (10µM) AGC AAT AGC ATC ACA AAT TTC ACA A SV40pA Reverse Primer

More information

EU Reference Laboratory for E. coli Department of Veterinary Public Health and Food Safety Unit of Foodborne Zoonoses Istituto Superiore di Sanità

EU Reference Laboratory for E. coli Department of Veterinary Public Health and Food Safety Unit of Foodborne Zoonoses Istituto Superiore di Sanità Identification and characterization of Verocytotoxin-producing Escherichia coli (VTEC) by Real Time PCR amplification of the main virulence genes and the genes associated with the serogroups mainly associated

More information

(A) Microarray analysis was performed on ATM and MDM isolated from 4 obese donors.

(A) Microarray analysis was performed on ATM and MDM isolated from 4 obese donors. Legends of supplemental figures and tables Figure 1: Overview of study design and results. (A) Microarray analysis was performed on ATM and MDM isolated from 4 obese donors. After raw data gene expression

More information

Taqman TCID50 for AAV Vector Infectious Titer Determination

Taqman TCID50 for AAV Vector Infectious Titer Determination Page 1 of 8 Purpose: To determine the concentration of infectious particles in an AAV vector sample. This process involves serial dilution of the vector in a TCID50 format and endpoint determination through

More information

Mutations and Genetic Variability. 1. What is occurring in the diagram below?

Mutations and Genetic Variability. 1. What is occurring in the diagram below? Mutations and Genetic Variability 1. What is occurring in the diagram below? A. Sister chromatids are separating. B. Alleles are independently assorting. C. Genes are replicating. D. Segments of DNA are

More information

Description: Molecular Biology Services and DNA Sequencing

Description: Molecular Biology Services and DNA Sequencing Description: Molecular Biology s and DNA Sequencing DNA Sequencing s Single Pass Sequencing Sequence data only, for plasmids or PCR products Plasmid DNA or PCR products Plasmid DNA: 20 100 ng/μl PCR Product:

More information

Table S1. Related to Figure 4

Table S1. Related to Figure 4 Table S1. Related to Figure 4 Final Diagnosis Age PMD Control Control 61 15 Control 67 6 Control 68 10 Control 49 15 AR-PD PD 62 15 PD 65 4 PD 52 18 PD 68 10 AR-PD cingulate cortex used for immunoblot

More information

NimbleGen SeqCap EZ Library SR User s Guide Version 3.0

NimbleGen SeqCap EZ Library SR User s Guide Version 3.0 NimbleGen SeqCap EZ Library SR User s Guide Version 3.0 For life science research only. Not for use in diagnostic procedures. Copyright 2011 Roche NimbleGen, Inc. All Rights Reserved. Editions Version

More information

Frozen-EZ Yeast Transformation II Catalog No. T2001

Frozen-EZ Yeast Transformation II Catalog No. T2001 INSTRUCTIONS Frozen-EZ Yeast Transformation II Catalog No. T2001 Highlights High transformation efficiency that yields approximately 10 5-10 6 transformants per μg plasmid DNA (circular). Frozen storage

More information

Next Generation Sequencing

Next Generation Sequencing Next Generation Sequencing 38. Informationsgespräch der Blutspendezentralefür Wien, Niederösterreich und Burgenland Österreichisches Rotes Kreuz 22. November 2014, Parkhotel Schönbrunn Die Zukunft hat

More information

First Strand cdna Synthesis

First Strand cdna Synthesis 380PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name First Strand cdna Synthesis (Cat. # 786 812) think proteins! think G-Biosciences

More information

SERVICES CATALOGUE WITH SUBMISSION GUIDELINES

SERVICES CATALOGUE WITH SUBMISSION GUIDELINES SERVICES CATALOGUE WITH SUBMISSION GUIDELINES 3921 Montgomery Road Cincinnati, Ohio 45212 513-841-2428 www.agctsequencing.com CONTENTS Welcome Dye Terminator Sequencing DNA Sequencing Services - Full Service

More information

Supplementary Information. Binding region and interaction properties of sulfoquinovosylacylglycerol (SQAG) with human

Supplementary Information. Binding region and interaction properties of sulfoquinovosylacylglycerol (SQAG) with human Supplementary Information Binding region and interaction properties of sulfoquinovosylacylglycerol (SQAG) with human vascular endothelial growth factor 165 revealed by biosensor based assays Yoichi Takakusagi

More information

Bacillus Subtilis Expression Vectors. Product Information and Instructions November 2005

Bacillus Subtilis Expression Vectors. Product Information and Instructions November 2005 Bacillus Subtilis Expression Vectors Product Information and Instructions November 2005 1 Content 1. Introduction... 3 2. The pht Vectors...4 2.1. Vector Map pht01...4 2.2. Vector Map pht43...5 2.3. Location

More information

The p53 MUTATION HANDBOOK

The p53 MUTATION HANDBOOK The p MUTATION HANDBOOK Version 1. /7 Thierry Soussi Christophe Béroud, Dalil Hamroun Jean Michel Rubio Nevado http://p/free.fr The p Mutation HandBook By T Soussi, J.M. Rubio-Nevado, D. Hamroun and C.

More information

Molecular analyses of EGFR: mutation and amplification detection

Molecular analyses of EGFR: mutation and amplification detection Molecular analyses of EGFR: mutation and amplification detection Petra Nederlof, Moleculaire Pathologie NKI Amsterdam Henrique Ruijter, Ivon Tielen, Lucie Boerrigter, Aafke Ariaens Outline presentation

More information

Wizard DNA Clean-Up System INSTRUCTIONS FOR USE OF PRODUCT A7280.

Wizard DNA Clean-Up System INSTRUCTIONS FOR USE OF PRODUCT A7280. Technical Bulletin Wizard DNA Clean-Up System INSTRUCTIONS FOR USE OF PRODUCT A7280. PRINTED IN USA. Revised 4/06 AF9TB141 0406TB141 Wizard DNA Clean-Up System All technical literature is available on

More information

Hands on Simulation of Mutation

Hands on Simulation of Mutation Hands on Simulation of Mutation Charlotte K. Omoto P.O. Box 644236 Washington State University Pullman, WA 99164-4236 omoto@wsu.edu ABSTRACT This exercise is a hands-on simulation of mutations and their

More information

Chromatin Immunoprecipitation

Chromatin Immunoprecipitation Chromatin Immunoprecipitation A) Prepare a yeast culture (see the Galactose Induction Protocol for details). 1) Start a small culture (e.g. 2 ml) in YEPD or selective media from a single colony. 2) Spin

More information

Title : Parallel DNA Synthesis : Two PCR product from one DNA template

Title : Parallel DNA Synthesis : Two PCR product from one DNA template Title : Parallel DNA Synthesis : Two PCR product from one DNA template Bhardwaj Vikash 1 and Sharma Kulbhushan 2 1 Email: vikashbhardwaj@ gmail.com 1 Current address: Government College Sector 14 Gurgaon,

More information

Cloning GFP into Mammalian cells

Cloning GFP into Mammalian cells Protocol for Cloning GFP into Mammalian cells Studiepraktik 2013 Molecular Biology and Molecular Medicine Aarhus University Produced by the instructors: Tobias Holm Bønnelykke, Rikke Mouridsen, Steffan

More information

Recombinant DNA Unit Exam

Recombinant DNA Unit Exam Recombinant DNA Unit Exam Question 1 Restriction enzymes are extensively used in molecular biology. Below are the recognition sites of two of these enzymes, BamHI and BclI. a) BamHI, cleaves after the

More information

MGC premier full length cdna and ORF clones

MGC premier full length cdna and ORF clones MGC premier full length cdna and ORF clones TCH1003, TCM1004, TCR1005, TCB1006, TCL1007, TCT1008, TCZ1009, TOH6003, TOM6004, TOZ6009, TCHS1003, TCMS1004, TCRS1005, TCBS1006, TCLS1007, TCTS1008 MGC premier

More information

Improved methods for site-directed mutagenesis using Gibson Assembly TM Master Mix

Improved methods for site-directed mutagenesis using Gibson Assembly TM Master Mix CLONING & MAPPING DNA CLONING DNA AMPLIFICATION & PCR EPIGENETICS RNA ANALYSIS Improved methods for site-directed mutagenesis using Gibson Assembly TM Master Mix LIBRARY PREP FOR NET GEN SEQUENCING PROTEIN

More information

Classic Immunoprecipitation

Classic Immunoprecipitation 292PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Classic Immunoprecipitation Utilizes Protein A/G Agarose for Antibody Binding (Cat.

More information

HighPure Maxi Plasmid Kit

HighPure Maxi Plasmid Kit HighPure Maxi Plasmid Kit For purification of high pure plasmid DNA with high yields www.tiangen.com PP120109 HighPure Maxi Plasmid Kit Kit Contents Storage Cat.no. DP116 Contents RNaseA (100 mg/ml) Buffer

More information

Genomic DNA Clean & Concentrator Catalog Nos. D4010 & D4011

Genomic DNA Clean & Concentrator Catalog Nos. D4010 & D4011 Page 0 INSTRUCTION MANUAL Catalog Nos. D4010 & D4011 Highlights Quick (5 minute) spin column recovery of large-sized DNA (e.g., genomic, mitochondrial, plasmid (BAC/PAC), viral, phage, (wga)dna, etc.)

More information

Application Guide... 2

Application Guide... 2 Protocol for GenomePlex Whole Genome Amplification from Formalin-Fixed Parrafin-Embedded (FFPE) tissue Application Guide... 2 I. Description... 2 II. Product Components... 2 III. Materials to be Supplied

More information

Angelo Peschiaroli, N. Valerio Dorrello, Daniele Guardavaccaro, Monica Venere, Thanos Halazonetis, Nicholas E. Sherman, and Michele Pagano

Angelo Peschiaroli, N. Valerio Dorrello, Daniele Guardavaccaro, Monica Venere, Thanos Halazonetis, Nicholas E. Sherman, and Michele Pagano Molecular Cell, Volume 23 Supplemental Data SCF βtrcp -Mediated Degradation of Claspin Regulates Recovery from the DNA Replication Checkpoint Response Angelo Peschiaroli, N. Valerio Dorrello, Daniele Guardavaccaro,

More information

Introduction to Perl Programming Input/Output, Regular Expressions, String Manipulation. Beginning Perl, Chap 4 6. Example 1

Introduction to Perl Programming Input/Output, Regular Expressions, String Manipulation. Beginning Perl, Chap 4 6. Example 1 Introduction to Perl Programming Input/Output, Regular Expressions, String Manipulation Beginning Perl, Chap 4 6 Example 1 #!/usr/bin/perl -w use strict; # version 1: my @nt = ('A', 'C', 'G', 'T'); for

More information

RIBOPROTECT. RNase Inhibitor RT33-020, RT33-100

RIBOPROTECT. RNase Inhibitor RT33-020, RT33-100 RIBOPROTECT RT33-020, RT33-100 RT33-020, RT33-100 RIBOPROTECT The RIBOPROTECT is a recombinant protein isolated and purified from Escherichia coli. It inhibits ribonuclease (RNase) activity of enzymes

More information

Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION

Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION Susan Carson Heather B. Miller D.Scott Witherow ELSEVIER AMSTERDAM BOSTON HEIDELBERG LONDON NEW YORK OXFORD PARIS SAN DIEGO SAN

More information

ptune Inducible Vector

ptune Inducible Vector ptune Inducible Vector Application Guide Table of Contents Package contents and Storage Conditions:...2 Related products:...2 Introduction...2 Figure 1. Schematic Diagrams of ptune Inducible vector...3

More information

BacReady TM Multiplex PCR System

BacReady TM Multiplex PCR System BacReady TM Multiplex PCR System Technical Manual No. 0191 Version 10112010 I Description.. 1 II Applications 2 III Key Features.. 2 IV Shipping and Storage. 2 V Simplified Procedures. 2 VI Detailed Experimental

More information

MultiSite Gateway Pro

MultiSite Gateway Pro MultiSite Gateway Pro Using Gateway Technology to simultaneously clone multiple DNA fragments Catalog nos. 12537-102, 12537-103, 12537-104, 12537-100 Version B 3 October 2006 25-0942 Design your MultiSite

More information

AxyPrep TM Mag PCR Clean-up Protocol

AxyPrep TM Mag PCR Clean-up Protocol AxyPrep TM Mag PCR Clean-up Protocol Intro The AxyPrep Mag PCR Clean-up kit utilizes a unique paramagnetic bead technology for rapid, high-throughput purification of PCR amplicons. Using this kit, PCR

More information

CLONING IN ESCHERICHIA COLI

CLONING IN ESCHERICHIA COLI CLONING IN ESCHERICHIA COLI Introduction: In this laboratory, you will carry out a simple cloning experiment in E. coli. Specifically, you will first create a recombinant DNA molecule by carrying out a

More information

In vitro analysis of pri-mirna processing. by Drosha-DGCR8 complex. (Narry Kim s lab)

In vitro analysis of pri-mirna processing. by Drosha-DGCR8 complex. (Narry Kim s lab) In vitro analysis of pri-mirna processing by Drosha-DGCR8 complex (Narry Kim s lab) 1-1. Preparation of radiolabeled pri-mirna transcript The RNA substrate for a cropping reaction can be prepared by in

More information

QUANTITATIVE RT-PCR. A = B (1+e) n. A=amplified products, B=input templates, n=cycle number, and e=amplification efficiency.

QUANTITATIVE RT-PCR. A = B (1+e) n. A=amplified products, B=input templates, n=cycle number, and e=amplification efficiency. QUANTITATIVE RT-PCR Application: Quantitative RT-PCR is used to quantify mrna in both relative and absolute terms. It can be applied for the quantification of mrna expressed from endogenous genes, and

More information

Stable Expression Clones and Auto-Induction for Protein Production in E. coli

Stable Expression Clones and Auto-Induction for Protein Production in E. coli Stable Expression Clones and Auto-Induction for Protein Production in E. coli F. William Studier Biosciences Department Brookhaven National Laboratory Upton, New York 11973-5000 studier@bnl.gov Running

More information

GENOTYPING ASSAYS AT ZIRC

GENOTYPING ASSAYS AT ZIRC GENOTYPING ASSAYS AT ZIRC A. READ THIS FIRST - DISCLAIMER Dear ZIRC user, We now provide detailed genotyping protocols for a number of zebrafish lines distributed by ZIRC. These protocols were developed

More information

restriction enzymes 350 Home R. Ward: Spring 2001

restriction enzymes 350 Home R. Ward: Spring 2001 restriction enzymes 350 Home Restriction Enzymes (endonucleases): molecular scissors that cut DNA Properties of widely used Type II restriction enzymes: recognize a single sequence of bases in dsdna, usually

More information

Molecular and Cell Biology Laboratory (BIOL-UA 223) Instructor: Ignatius Tan Phone: 212-998-8295 Office: 764 Brown Email: ignatius.tan@nyu.

Molecular and Cell Biology Laboratory (BIOL-UA 223) Instructor: Ignatius Tan Phone: 212-998-8295 Office: 764 Brown Email: ignatius.tan@nyu. Molecular and Cell Biology Laboratory (BIOL-UA 223) Instructor: Ignatius Tan Phone: 212-998-8295 Office: 764 Brown Email: ignatius.tan@nyu.edu Course Hours: Section 1: Mon: 12:30-3:15 Section 2: Wed: 12:30-3:15

More information

INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE Q5B

INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE Q5B INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE ICH HARMONISED TRIPARTITE GUIDELINE QUALITY OF BIOTECHNOLOGICAL PRODUCTS: ANALYSIS

More information

APOT - Assay. Protocol for HPV16 and 18. Amplification of Papilloma Virus Oncogene Transcripts HPV. E6 E7 E1 Zelluläre DNA poly(a)

APOT - Assay. Protocol for HPV16 and 18. Amplification of Papilloma Virus Oncogene Transcripts HPV. E6 E7 E1 Zelluläre DNA poly(a) E5 E2 E1 APOT - Assay Amplification of Papilloma Virus Oncogene Transcripts URR E6 E7 L1 HPV L2 E4 E6 E7 E1 Zelluläre DNA poly(a) Protocol for HPV16 and 18 Brief summary of the APOT assay Fig.1A shows

More information

Mutation. Mutation provides raw material to evolution. Different kinds of mutations have different effects

Mutation. Mutation provides raw material to evolution. Different kinds of mutations have different effects Mutation Mutation provides raw material to evolution Different kinds of mutations have different effects Mutational Processes Point mutation single nucleotide changes coding changes (missense mutations)

More information

PrimeSTAR HS DNA Polymerase

PrimeSTAR HS DNA Polymerase Cat. # R010A For Research Use PrimeSTAR HS DNA Polymerase Product Manual Table of Contents I. Description...3 II. III. IV. Components...3 Storage...3 Features...3 V. General Composition of PCR Reaction

More information

June 09, 2009 Random Mutagenesis

June 09, 2009 Random Mutagenesis Why Mutagenesis? Analysis of protein function June 09, 2009 Random Mutagenesis Analysis of protein structure Protein engineering Analysis of structure-function relationship Analysis of the catalytic center

More information

Molecular Cloning, Product Brochure

Molecular Cloning, Product Brochure , Product Brochure Interest in any of the products, request or order them at Bio-Connect. Bio-Connect B.V. T NL +31 (0)26 326 44 50 T BE +32 (0)2 503 03 48 Begonialaan 3a F NL +31 (0)26 326 44 51 F BE

More information

GRS Plasmid Purification Kit Transfection Grade GK73.0002 (2 MaxiPreps)

GRS Plasmid Purification Kit Transfection Grade GK73.0002 (2 MaxiPreps) 1 GRS Plasmid Purification Kit Transfection Grade GK73.0002 (2 MaxiPreps) (FOR RESEARCH ONLY) Sample : Expected Yield : Endotoxin: Format : Operation Time : Elution Volume : 50-400 ml of cultured bacterial

More information

ChIP TROUBLESHOOTING TIPS

ChIP TROUBLESHOOTING TIPS ChIP TROUBLESHOOTING TIPS Creative Diagnostics Abstract ChIP dissects the spatial and temporal dynamics of the interactions between chromatin and its associated factors CD Creative Diagnostics info@creative-

More information

TA Cloning Kit. Version V 7 April 2004 25-0024. Catalog nos. K2000-01, K2000-40, K2020-20, K2020-40, K2030-01 K2030-40, K2040-01, K2040-40

TA Cloning Kit. Version V 7 April 2004 25-0024. Catalog nos. K2000-01, K2000-40, K2020-20, K2020-40, K2030-01 K2030-40, K2040-01, K2040-40 TA Cloning Kit Version V 7 April 2004 25-0024 TA Cloning Kit Catalog nos. K2000-01, K2000-40, K2020-20, K2020-40, K2030-01 K2030-40, K2040-01, K2040-40 ii Table of Contents Table of Contents...iii Important

More information

Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity

Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity Today you analyze the results of your bacterial transformation from last week and determine the efficiency

More information

SUPPLEMENTARY FIGURES

SUPPLEMENTARY FIGURES SUPPLEMENTARY FIGURES Fig. S1: Effect of ISO- and TAC-treatments on the biosynthesis of FAS-II elongation products in M. tb H37Ra. LC/MS chromatograms showing a decrease in products with elemental compositions

More information

How To Use An Enzymatics Spark Dna Sample Prep Kit For Ion Torrent

How To Use An Enzymatics Spark Dna Sample Prep Kit For Ion Torrent SPARK DNA Sample Prep Kit Ion Torrent (SPK0002-V08) Frequently Asked Questions Under what circumstances would I use SPARK DNA Sample Prep Kit for Ion Torrent? Enzymatics SPARK DNA Sample Prep Kit for Ion

More information

TECHNICAL BULLETIN. HIS-Select Nickel Affinity Gel. Catalog Number P6611 Storage Temperature 2 8 C

TECHNICAL BULLETIN. HIS-Select Nickel Affinity Gel. Catalog Number P6611 Storage Temperature 2 8 C HIS-Select Nickel Affinity Gel Catalog Number P6611 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description HIS-Select Nickel Affinity Gel is an immobilized metalion affinity chromatography (IMAC)

More information

The Awesome Power of Yeast Genetics: Spontaneous and Induced Mutagenesis and Complementation Analysis using Saccharomyces cerevisiae.

The Awesome Power of Yeast Genetics: Spontaneous and Induced Mutagenesis and Complementation Analysis using Saccharomyces cerevisiae. The Awesome Power of Yeast Genetics: Spontaneous and Induced Mutagenesis and Complementation Analysis using Saccharomyces cerevisiae. Mutations occur as a consequence of normal cellular physiology and

More information

THE His Tag Antibody, mab, Mouse

THE His Tag Antibody, mab, Mouse THE His Tag Antibody, mab, Mouse Cat. No. A00186 Technical Manual No. TM0243 Update date 01052011 I Description.... 1 II Key Features. 2 III Storage 2 IV Applications.... 2 V Examples - ELISA..... 2 VI

More information

Recombinant Protein Expression and Purification from E. coli

Recombinant Protein Expression and Purification from E. coli Recombinant Protein Expression and Purification from E. coli Many different biochemistry projects require recombinant protein. This is a simple method for creating crude bacterial lysate of IPTG inducible

More information

His GraviTrap. GE Healthcare. Operation

His GraviTrap. GE Healthcare. Operation GE Healthcare Data File 11-0036-90 AB Affinity purification His GraviTrap His GraviTrap is a prepacked, single-use column for purification of histidine-tagged proteins by immobilized metal affinity chromatography

More information

High deleterious genomic mutation rate in stationary phase of Escherichia coli

High deleterious genomic mutation rate in stationary phase of Escherichia coli Loewe, L et al. (2003) "High deleterious genomic mutation rate in stationary phase of Escherichia coli" 1 High deleterious genomic mutation rate in stationary phase of Escherichia coli Laurence Loewe,

More information

Creating Standard Curves with Genomic DNA or Plasmid DNA Templates for Use in Quantitative PCR

Creating Standard Curves with Genomic DNA or Plasmid DNA Templates for Use in Quantitative PCR Creating Standard Curves with Genomic DNA or Plasmid DNA Templates for Use in Quantitative PCR Overview Genomic DNA (gdna) and plasmids containing cloned target sequences are commonly used as standards

More information

MEF Nucleofector Kit 1 and 2

MEF Nucleofector Kit 1 and 2 page 1 of 7 MEF Nucleofector Kit 1 and 2 for Mouse Embryonic Fibroblasts (MEF) MEF display significant phenotypic variations which depend on the strain, the genetic background of the they are isolated

More information

Transformation Protocol

Transformation Protocol To make Glycerol Stocks of Plasmids ** To be done in the hood and use RNase/DNase free tips** 1. In a 10 ml sterile tube add 3 ml autoclaved LB broth and 1.5 ul antibiotic (@ 100 ug/ul) or 3 ul antibiotic

More information

Cloning Blunt-End Pfu DNA Polymerase- Generated PCR Fragments into pgem -T Vector Systems

Cloning Blunt-End Pfu DNA Polymerase- Generated PCR Fragments into pgem -T Vector Systems Promega Notes Number 71, 1999, p. 10 Blunt-End Pfu DNA Polymerase- Generated PCR Fragments into pgem -T Vector Systems By Kimberly Knoche, Ph.D., and Dan Kephart, Ph.D. Promega Corporation Corresponding

More information

European Medicines Agency

European Medicines Agency European Medicines Agency July 1996 CPMP/ICH/139/95 ICH Topic Q 5 B Quality of Biotechnological Products: Analysis of the Expression Construct in Cell Lines Used for Production of r-dna Derived Protein

More information

BUFFERS and MEDIAS Coomassie Blue Staining Solution Coomassie blue Destaining Solution DMEM Normal Cell Culture Media

BUFFERS and MEDIAS Coomassie Blue Staining Solution Coomassie blue Destaining Solution DMEM Normal Cell Culture Media BUFFERS and MEDIAS Coomassie Blue Staining Solution 2 g Coomassie Blue 2 L Methanol or Ethanol * 1.6 L 400 ml Glacial acetic acid *If you will be microwaving the gel in staining solution for rapid staining

More information

pbad/his A, B, and C pbad/myc-his A, B, and C

pbad/his A, B, and C pbad/myc-his A, B, and C pbad/his A, B, and C pbad/myc-his A, B, and C Vectors for Dose-Dependent Expression of Recombinant Proteins Containing N- or C-Terminal 6 His Tags in E. coli Catalog nos. V430-01, V440-01 Version J 29

More information

Design of conditional gene targeting vectors - a recombineering approach

Design of conditional gene targeting vectors - a recombineering approach Recombineering protocol #4 Design of conditional gene targeting vectors - a recombineering approach Søren Warming, Ph.D. The purpose of this protocol is to help you in the gene targeting vector design

More information

inhibition of mitosis

inhibition of mitosis The EMBO Journal vol.13 no.2 pp.425-434, 1994 cdt 1 is an essential target of the Cdc 1 O/Sct 1 transcription factor: requirement for DNA replication and inhibition of mitosis Johannes F.X.Hofmann and

More information

Rapid GST Inclusion Body Solubilization and Renaturation Kit

Rapid GST Inclusion Body Solubilization and Renaturation Kit Product Manual Rapid GST Inclusion Body Solubilization and Renaturation Kit Catalog Number AKR-110 FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Bacteria are widely used for His

More information

Irina V Nesterova, Cecily A. Bennett, S. Sibel Erdem, Robert P. Hammer, Prescott L. Deininger, and Steven A. Soper

Irina V Nesterova, Cecily A. Bennett, S. Sibel Erdem, Robert P. Hammer, Prescott L. Deininger, and Steven A. Soper ear-ir Single Fluorophore Quenching System Based on Phthalocyanine (Pc) Aggregation and its Application for Monitoring Inhibitor/Activator Action on a Therapeutic Target: L1-E Irina V esterova, Cecily

More information

Five-minute cloning of Taq polymerase-amplified PCR products

Five-minute cloning of Taq polymerase-amplified PCR products TOPO TA Cloning Version R 8 April 2004 25-0184 TOPO TA Cloning Five-minute cloning of Taq polymerase-amplified PCR products Catalog nos. K4500-01, K4500-40, K4510-20, K4520-01, K4520-40, K4550-01, K4550-40,

More information

Bio 102 Practice Problems Recombinant DNA and Biotechnology

Bio 102 Practice Problems Recombinant DNA and Biotechnology Bio 102 Practice Problems Recombinant DNA and Biotechnology Multiple choice: Unless otherwise directed, circle the one best answer: 1. Which of the following DNA sequences could be the recognition site

More information

Mouse ES Cell Nucleofector Kit

Mouse ES Cell Nucleofector Kit page 1 of 7 Mouse ES Cell Nucleofector Kit for Mouse Embryonic Stem Cells Cell type Origin Cells derived from mouse blastocysts. Morphology Round cells growing in clumps. Important remarks! 1. This protocol

More information

Molecular Biology. Yeast Transformation. Yeast Plasmids. Gene Disruption, tagging. Cloning by Complementation. Epistasis

Molecular Biology. Yeast Transformation. Yeast Plasmids. Gene Disruption, tagging. Cloning by Complementation. Epistasis Molecular Biology Yeast Transformation Yeast Plasmids Gene Disruption, tagging Cloning by Complementation Epistasis Transformation Transformation: introduction of DNA 1978, ca 1000x less efficient than

More information

NIH Mammalian Gene Collection (MGC)

NIH Mammalian Gene Collection (MGC) USER GUIDE NIH Mammalian Gene Collection (MGC) Catalog number FL1002 Revision date 28 November 2011 Publication Part number 25-0610 MAN0000351 For Research Use Only. Not for diagnostic procedures. ii Table

More information

How To Clone Into Pcdna 3.1/V5-His

How To Clone Into Pcdna 3.1/V5-His pcdna 3.1/V5-His A, B, and C Catalog no. V810-20 Rev. date: 09 November 2010 Manual part no. 28-0141 MAN0000645 User Manual ii Contents Contents and Storage... iv Methods... 1 Cloning into pcdna 3.1/V5-His

More information

50 g 650 L. *Average yields will vary depending upon a number of factors including type of phage, growth conditions used and developmental stage.

50 g 650 L. *Average yields will vary depending upon a number of factors including type of phage, growth conditions used and developmental stage. 3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com Phage DNA Isolation Kit Product # 46800, 46850 Product Insert

More information

Chromatin Immunoprecipitation (ChIP)

Chromatin Immunoprecipitation (ChIP) Chromatin Immunoprecipitation (ChIP) Day 1 A) DNA shearing 1. Samples Dissect tissue (One Mouse OBs) of interest and transfer to an eppendorf containing 0.5 ml of dissecting media (on ice) or PBS but without

More information

Rapid and Simple Amplification of Genomic DNA Sequences Flanking Transposon

Rapid and Simple Amplification of Genomic DNA Sequences Flanking Transposon MICROBIOLOGY INDONESIA, April 2007, p 1-4 Volume. 1, Number 1 ISSN 1978-3477 Rapid and Simple Amplification of Genomic DNA Sequences Flanking Transposon ARIS TRI WAHYUDI Department of Biology, Institut

More information

MGC premier Expression-Ready cdna clones TCH1103, TCM1104, TCR1105, TCB1106, TCH1203, TCM1204, TCR1205, TCB1206, TCH1303, TCM1304, TCR1305

MGC premier Expression-Ready cdna clones TCH1103, TCM1104, TCR1105, TCB1106, TCH1203, TCM1204, TCR1205, TCB1206, TCH1303, TCM1304, TCR1305 MGC premier Expression-Ready cdna clones TCH1103, TCM1104, TCR1105, TCB1106, TCH1203, TCM1204, TCR1205, TCB1206, TCH1303, TCM1304, TCR1305 The MGC premier Expression-Ready collection has the high quality,

More information

DNA Core Facility: DNA Sequencing Guide

DNA Core Facility: DNA Sequencing Guide DNA Core Facility: DNA Sequencing Guide University of Missouri-Columbia 216 Life Sciences Center Columbia, MO 65211 http://biotech.missouri.edu/dnacore/ Table of Contents 1. Evaluating Sequencing Data..

More information