Creating pentr vectors by BP reaction

Size: px
Start display at page:

Download "Creating pentr vectors by BP reaction"

Transcription

1 Creating pentr vectors by BP reaction Tanya Lepikhova and Rafael Martinez Overview: 1. Design primers to add the attb sites to gene of interest 2. Perform PCR with a high fidelity DNA polymerase 3. Purify PCR product 4. Perform a BP reaction of the gene into pdonr Transform the BP reaction into DH5-alpha 6. Verify contructs Example: In the following example we are going to generate a pentr vector inserting a gene of 2500 pb by BP reaction into pdonr221 kanamycin resistant (Invitrogen ). 1. Design primers to add the attb sites to gene of interest Take in count that if you want to express your gene in Eukaryotic systems a Kozak consensus sequence is needed. If you want to express the gene in Procaryotic systems add a Shine-Dalgarno consensus sequence. If wished both sequences can be added to the primers. The primers for a BP reaction must contain: 1. Four guanine (G) residues at the 5 end followed by 2. The 25 bp attb1 site followed by 3. At least bp of template- or gene-specific sequences C-terminal fusion proteins. If you want to express your protein with a C-terminal fusion tag do not add a stop codon. N-terminal fusion proteins. To express protein with N-teminal tag be sure that the two codons for Lys (AAA AAA) are in frame with start codon (see next page). This primer will work also for C-terminal fusion proteins. For more details check the Invitrogen Manual. Some examples are provided in the following page:

2 Primer design examples for BP reaction

3 2. Perform PCR with a high fidelity DNA polymerase Use a high fidelity enzyme to generate a PCR product i.e Phusion (Finnzymes F-530S) or TaKaRa Ex Taq (TaKaRa RR006A). Perform 2-4 reactions with a reaction volume of 50µl each. 3. Purify PCR product Check in agarose gel that the PCR product is of the expected size. If everything is correct proceed to clean the rest of the PCR product with a spin column (i.e Qiagen). Continue with the BP reaction. Note: if the BP reaction does not work repeat the PCR reaction and clean the product by cutting the band from an agarose gel. 4. Perform a BP reaction of the gene into pdonr221 To reduce background in the BP reaction linearize the pdonr with EcoRI. This enzyme cuts right in the middle of the gateway cassette. Inactivate the enzyme incubating at 65 C. Calculate 50 fmol and 100 fmol of your PCR product by using the following formula: where N is the size of the DNA in bp. Some examples are shown in the following table: Note: 50fmol of pdonr221 are 150ng

4 BP reaction Material: Gene of interest with att sites added by PCR Linearized pdonr221 (Invitrogen ) BP clonase (Invitrogen ) TE 1x Buffer ph 8.0 Proteinase K (included in the BP clonase kit) Positive control pexp7-let (included in the BP clonase kit) BP Reaction: Perform the BP reaction testing 50 and 100fmol of PCR product and pdonr221. Also include pexp7- let as a positive control. A typical reaction is as the following: 5. Transform the BP reaction into DH5-alpha Material: Method: Library Efficiency DH5α (Invitrogen ) BP reaction SOC medium without antibiotics LB agar plates with + Kanamycin 50µg/ml L-Spreaders Thermal Block at 42ºC. 1. Thaw the bacteria on ice (10-15min). Keep the bacteria on ice at any time!. Aliquot 20µl for each reaction in 1.5 ml eppendorf tubes. If there is still some bacteria left in the vial bring it back as soon as possible to -80 C.

5 2. Add 2µl from the BP reaction to 20µl competent cells and stir gently with the tip (do not pipette up and down!). Incubate 30min on ice. 3. Heat shock at 42 C for 30seconds. 4. Incubate on ice 3min. 5. Add 250µl SOC medium to the transformed competent cells and incubate at 37 C with shaking at 230rpm for 1hr. 6. While incubating label the LB-agar plates on the agar-side with: Gene/pENTR; Type of cell; Name and Date. 7. Spread 150µl of the transformed bacteria on agar plates with Kanamycin 50µg/ml. With a "L" tool spread uniformly all over the agar. Let the plate stand 5 min with the agar-side down to let the liquid be absorb. 8. Incubate overnight at 37 C with agar-side up. If everything is perfect colonies of around 0.9 mm will appear all over the plate. After incubation, plates can be store at 4 C for 1 month. TIP. Keep the tube from step 5 at 4ºC. In case there are too many colonies spread 50µl next day. TIP. Spreading 150 µl of bacteria in step 7 is a general rule. If there are difficult clones then spread the whole 250µl if necessary. 6. Verifying constructs 1. Pick up single colonies and incubate them in 5 ml LB medium with kanamycin 50µg/ml. Incubate overnight at 37 C with shaking at 230rpm. 2. Perform minipreps with 2 ml, store the rest of culture at 4ºC. 3. Verify your insert with enzyme digestion. 4. If everything is ok then prepare a glycerol stock (200µl Glycerol + 800µl media) and store at -80ºC. Digestion analysis with PstI and EcoRI of a pentr221 construct created by BP reaction of a PCR product of 2500pb into pdonr221. According with the expected digestion pattern samples on wells 3 and 4 have the right plasmid.

6 References: Cloning/GatewayC-Misc/Protocols.html

Conversion of plasmids into Gateway compatible cloning

Conversion of plasmids into Gateway compatible cloning Conversion of plasmids into Gateway compatible cloning Rafael Martinez 14072011 Overview: 1. Select the right Gateway cassette (A, B or C). 2. Design primers to amplify the right Gateway cassette from

More information

Cold Fusion Cloning Kit. Cat. #s MC100A-1, MC101A-1. User Manual

Cold Fusion Cloning Kit. Cat. #s MC100A-1, MC101A-1. User Manual Fusion Cloning technology Cold Fusion Cloning Kit Store the master mixture and positive controls at -20 C Store the competent cells at -80 C. (ver. 120909) A limited-use label license covers this product.

More information

Polymerase Chain Reaction

Polymerase Chain Reaction Polymerase Chain Reaction Amplify your insert or verify its presence 3H Taq platinum PCR mix primers Ultrapure Water PCR tubes PCR machine A. Insert amplification For insert amplification, use the Taq

More information

pgm-t Cloning Kit Cat. # : GVT202 Size : 20 Reactions Store at -20

pgm-t Cloning Kit Cat. # : GVT202 Size : 20 Reactions Store at -20 pgm-t Cloning Kit Cat. # : GVT202 Size : 20 Reactions Store at -20 1 Kit Contents Contents pgm-t Cloning Kit pgm-t Vector (50 ng/μl) 20 μl T4 DNA Ligase (3 U/μl) 20 μl 10X T4 DNA Ligation Buffer 30 μl

More information

pgm-t Cloning Kit For direct cloning of PCR products generated by Taq DNA polymerases For research use only Cat. # : GVT202 Size : 20 Reactions

pgm-t Cloning Kit For direct cloning of PCR products generated by Taq DNA polymerases For research use only Cat. # : GVT202 Size : 20 Reactions pgm-t Cloning Kit For direct cloning of PCR products generated by Taq DNA polymerases Cat. # : GVT202 Size : 20 Reactions Store at -20 For research use only 1 pgm-t Cloning Kit Cat. No.: GVT202 Kit Contents

More information

peco TM -T7-nGST, Eco cloning Kit User Manual (Patent pending)

peco TM -T7-nGST, Eco cloning Kit User Manual (Patent pending) peco TM -T7-nGST, Eco cloning Kit User Manual (Patent pending) Cloning PCR products for E Coli expression of N-term GST-tagged protein Cat# Contents Amounts Application IC-1004 peco-t7-ngst vector built-in

More information

Data Sheet Quick PCR Cloning Kit

Data Sheet Quick PCR Cloning Kit Data Sheet Quick PCR Cloning Kit 6044 Cornerstone Ct. West, Ste. E DESCRIPTION: The Quick PCR Cloning Kit is a simple and highly efficient method to insert any gene or DNA fragment into a vector, without

More information

Gateway Cloning Protocol (Clough Lab Edition) This document is a modification of the Gateway cloning protocol developed by Manju in Chris Taylor's lab

Gateway Cloning Protocol (Clough Lab Edition) This document is a modification of the Gateway cloning protocol developed by Manju in Chris Taylor's lab Gateway Cloning Protocol (Clough Lab Edition) This document is a modification of the Gateway cloning protocol developed by Manju in Chris Taylor's lab With the Gateway cloning system, a PCR fragment is

More information

Ready_to_use Fast Seamless Cloning Kit. User Manual

Ready_to_use Fast Seamless Cloning Kit. User Manual For general laboratory use. Not for use in diagnostic procedures. FOR IN VITRO USE ONLY. Ready_to_use Fast Seamless Cloning Kit User Manual 1 / 6 Tel: 021-58975266 Fax: 021-50800270 Email:tech@dogene.com

More information

Lab Book igem Stockholm Lysostaphin. Week 6

Lab Book igem Stockholm Lysostaphin. Week 6 Lysostaphin Week 6 Summarized below are the experiments conducted this week in chronological order. Click on the experiment name to view it. To go back to this summary, click Summary in the footer. Summary

More information

Hetero-Stagger PCR Cloning Kit

Hetero-Stagger PCR Cloning Kit Product Name: Code No: Size: DynaExpress Hetero-Stagger PCR Cloning Kit DS150 20 reactions Kit Components: Box 1 (-20 ) phst-1 Vector, linearized Annealing Buffer Ligase Mixture phst Forward Sequence Primer

More information

Perform HiFi PCR Purify PCR-mixture using PCR-clean up kit and determine concentration

Perform HiFi PCR Purify PCR-mixture using PCR-clean up kit and determine concentration IGEM EXPERIMENT 2 CONSTRUCTION OF PSB#X#-T7CCDB Strategy Cloning Flow Chart (using restriction/ligation): 1. In silico design of cloning Design a cloning strategy (in case of insert/backbone): o Using

More information

HE Swift Cloning Kit

HE Swift Cloning Kit HE Swift Cloning Kit For high-efficient cloning of PCR products either blunt or sticky-end Kit Contents Contents VTT-BB05 phe Vector (35 ng/µl) 20 µl T4 DNA Ligase (3 U/µl) 20 µl 2 Reaction Buffer 100

More information

Cat # Box1 Box2. DH5a Competent E. coli cells CCK-20 (20 rxns) 40 µl 40 µl 50 µl x 20 tubes. Choo-Choo Cloning TM Enzyme Mix

Cat # Box1 Box2. DH5a Competent E. coli cells CCK-20 (20 rxns) 40 µl 40 µl 50 µl x 20 tubes. Choo-Choo Cloning TM Enzyme Mix Molecular Cloning Laboratories User Manual Version 3.3 Product name: Choo-Choo Cloning Kits Cat #: CCK-10, CCK-20, CCK-096, CCK-384 Description: Choo-Choo Cloning is a highly efficient directional PCR

More information

GenBuilder TM Plus Cloning Kit User Manual

GenBuilder TM Plus Cloning Kit User Manual GenBuilder TM Plus Cloning Kit User Manual Cat. No. L00744 Version 11242017 Ⅰ. Introduction... 2 I.1 Product Information... 2 I.2 Kit Contents and Storage... 2 I.3 GenBuilder Cloning Kit Workflow... 2

More information

GenBuilder TM Plus Cloning Kit User Manual

GenBuilder TM Plus Cloning Kit User Manual GenBuilder TM Plus Cloning Kit User Manual Cat.no L00744 Version 11242017 Ⅰ. Introduction... 2 I.1 Product Information... 2 I.2 Kit Contents and Storage... 2 I.3 GenBuilder Cloning Kit Workflow... 2 Ⅱ.

More information

Cloning Multiple Gateway Reaction

Cloning Multiple Gateway Reaction Cloning Multiple Gateway Reaction by A. Untergasser (contact address and download at www.untergasser.de/lab) Version: 1.0 - Print Version (.PDF) ATTENTION: This is not something what is done with low quality

More information

Protocols. We used a buffer mix with polymerase (either Mango Mix (forhandler) or 2x High Fidelity) and the appropriate primers and template DNA.

Protocols. We used a buffer mix with polymerase (either Mango Mix (forhandler) or 2x High Fidelity) and the appropriate primers and template DNA. Protocols PCRs Colony PCRs We used a buffer mix with polymerase (either Mango Mix (forhandler) or 2x High Fidelity) and the appropriate primers and template DNA. For a 10 µl reaction 2x Mango Mix/2x High

More information

Generation of Gene Targeting Vectors using Recombineering - Small scale (single tube) protocol

Generation of Gene Targeting Vectors using Recombineering - Small scale (single tube) protocol Generation of Gene Targeting Vectors using Recombineering - Small scale (single tube) protocol Reagent information All TSA bacterial liquid cultures are grown in: 1xTerrific Broth (TB) supplemented with

More information

Rapid amplification of cdna ends (RACE)

Rapid amplification of cdna ends (RACE) Rapid amplification of cdna ends (RACE) Rapid amplification of cdna ends (RACE) is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell. RACE

More information

Designing and creating your gene knockout Background The rada gene was identified as a gene, that when mutated, caused cells to become hypersensitive

Designing and creating your gene knockout Background The rada gene was identified as a gene, that when mutated, caused cells to become hypersensitive Designing and creating your gene knockout Background The rada gene was identified as a gene, that when mutated, caused cells to become hypersensitive to ionizing radiation. However, why these mutants are

More information

VDL101.3 CLONING TRANSGENE INTO pad5f35

VDL101.3 CLONING TRANSGENE INTO pad5f35 Purpose 1.1. The purpose of this protocol is to transfer a transgene from the pshuttlex plasmid to pad5/f35. 1.2. The starting material is 10 μg plasmid DNA. 1.3. This procedure is routinely performed

More information

GenBuilder TM Cloning Kit User Manual

GenBuilder TM Cloning Kit User Manual GenBuilder TM Cloning Kit User Manual Cat.no L00701 Version 11242017 Ⅰ. Introduction... 2 I.1 Product Information... 2 I.2 Kit Contents and Storage... 2 I.3 GenBuilder Cloning Kit Workflow... 2 Ⅱ. DNA

More information

Application of Molecular Biology tools for cloning of a foreign gene

Application of Molecular Biology tools for cloning of a foreign gene IFM/Kemi Linköpings Universitet September 2013/LGM Labmanual Project course Application of Molecular Biology tools for cloning of a foreign gene Table of contents Introduction... 3 Amplification of a gene

More information

BP-reaction (recombination of PCR products into entry vector) Created on: 06/01/10 Version: 1.1 No.: 1 Page 1 of 6

BP-reaction (recombination of PCR products into entry vector) Created on: 06/01/10 Version: 1.1 No.: 1 Page 1 of 6 Created on: 06/01/10 Version: 1.1 No.: 1 Page 1 of 6 1. Background This reaction generates bacteria that contain recombinant Gateway-compatible entry plasmid. The PCRamplified ORFs (SOPs ORF-PCR 1 (two

More information

Table of contents. I. Description II. Kit Components III. Reagents and Instruments Required IV. Storage V. Protocols...

Table of contents. I. Description II. Kit Components III. Reagents and Instruments Required IV. Storage V. Protocols... Table of contents I. Description... 2 II. Kit Components... 2 III. Reagents and Instruments Required... 2 IV. Storage... 2 V. Protocols... 3 VI-1. Procedure... 3 VI-2. Note... 4 VI. Control experiment...

More information

Figure 1. Map of cloning vector pgem T-Easy (bacterial plasmid DNA)

Figure 1. Map of cloning vector pgem T-Easy (bacterial plasmid DNA) Texas A&M University-Corpus Christi CHEM4402 Biochemistry II Laboratory Laboratory 6: Ligation & Bacterial Transformation (Bring your text and laptop to class if you wish to work on your assignment during

More information

Recombineering Manual

Recombineering Manual Recombineering Manual Anthony Popkie The Phiel Laboratory The Research Institute at Nationwide Childrenʼs Hospital 1 BAC Transformation BACs may be transformed into either DY380, EL250 or EL350 cells.

More information

Updated August well High-multiplexing Tagmentation and Amplification Robyn Tanny August Company Kit Catalog Number.

Updated August well High-multiplexing Tagmentation and Amplification Robyn Tanny August Company Kit Catalog Number. 96-well High-multiplexing Tagmentation and Amplification Robyn Tanny August 2015 This protocol uses the following purchased reagents: Company Kit Catalog Number Illumina Nextera DNA Sample Preparation

More information

In order to make our construct pah12 (Mlra) and pah05 (venus) biobrick compatitable PCR was conducted. Following PCR mixes were prepared

In order to make our construct pah12 (Mlra) and pah05 (venus) biobrick compatitable PCR was conducted. Following PCR mixes were prepared BioBrick cloning Project: BioBricks Authors: Antti Koistinen Dates: 2016-09-15 to 2016-10-06 THURSDAY, 9/15 In order to make our construct pah12 (Mlra) and pah05 (venus) biobrick compatitable PCR was conducted.

More information

NZYGene Synthesis kit

NZYGene Synthesis kit Kit components Component Concentration Amount NZYGene Synthesis kit Catalogue number: MB33901, 10 reactions GS DNA Polymerase 1U/ μl 30 μl Reaction Buffer for GS DNA Polymerase 10 150 μl dntp mix 2 mm

More information

Gibson Assembly. igem TU/e 2015 Biomedical Engineering

Gibson Assembly. igem TU/e 2015 Biomedical Engineering igem TU/e 2015 Biomedical Engineering Eindhoven University of Technology Room: Ceres 0.04 Den Dolech 2, 5612 AZ Eindhoven The Netherlands Tel. no. +31 50 247 55 59 2015.igem.org/Team:TU_Eindhoven Gibson

More information

KOD -Plus- Mutagenesis Kit

KOD -Plus- Mutagenesis Kit Instruction manual KOD -Plus- Mutagenesis Kit 0811 F0936K KOD -Plus- Mutagenesis Kit SMK-101 20 reactions Store at -20 C Contents [1] Introduction [2] Flow chart [3] Components [4] Notes [5] Protocol 1.

More information

InterLab Study: Plasmid amplification

InterLab Study: Plasmid amplification igem TU/e 2015 Biomedical Engineering Eindhoven University of Technology Room: Ceres 0.04 Den Dolech 2, 5612 AZ Eindhoven The Netherlands Tel. no. +31 50 247 55 59 2015.igem.org/Team:TU_Eindhoven InterLab

More information

Journal of Experimental Microbiology and Immunology (JEMI) Vol. 6:20-25 Copyright December 2004, M&I UBC

Journal of Experimental Microbiology and Immunology (JEMI) Vol. 6:20-25 Copyright December 2004, M&I UBC Preparing Plasmid Constructs to Investigate the Characteristics of Thiol Reductase and Flavin Reductase With Regard to Solubilizing Insoluble Proteinase Inhibitor 2 in Bacterial Protein Overexpression

More information

Subcloning 1. Overview 2. Method

Subcloning 1. Overview 2. Method Subcloning 1. Overview: 1.1. Digest plasmids for vector + insert 1.2. CIP treat vector 1.3. Gel purify vector + insert 1.4. Ligate 1.5. Transform bacteria with ligation reactions 1.6. Qiagen or Eppendorf

More information

mrnaexpress mrna Synthesis Kit Cat. #MR-KIT-1

mrnaexpress mrna Synthesis Kit Cat. #MR-KIT-1 mrnaexpress mrna Synthesis Kit Cat. #MR-KIT-1 User Manual Check Individual Components for Storage conditions ver. 2-070918 A limited-use label license covers this product. By use of this product, you accept

More information

igem LMU- Munich Munich Cloning procedure

igem LMU- Munich Munich Cloning procedure igem LM- Munich 2014 - igem@bio.lmu.de - http://2014.igem.org/team:lm- Munich Cloning procedure Overview of a cloning procedure: 1. Digestion of a PCR product ( insert) and a vector 2. Dephosphorylation

More information

Ligation Independent Cloning (LIC) Procedure

Ligation Independent Cloning (LIC) Procedure Ligation Independent Cloning (LIC) Procedure Ligation Independent Cloning (LIC) LIC cloning allows insertion of DNA fragments without using restriction enzymes into specific vectors containing engineered

More information

THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY. STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 084 MOD: 1st Issue Page: 1 of 11

THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY. STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 084 MOD: 1st Issue Page: 1 of 11 Page: 1 of 11 1. Risk Assessment: This Risk Assessment is to be used as a general guide and as such, cannot accommodate all the varying factors that may be encountered when using this procedure. Therefore,

More information

Transformation (The method of CaCl 2 )

Transformation (The method of CaCl 2 ) PROTOCOLS E. coli Transformation (The method of CaCl 2 ) Ligation PRODUCTION competent cells of E. COLI. (Rubidium cells). Gel DNA Recovery Kit Electroporation Digestiones Plasmid purification (The method

More information

Molecular Techniques Third-year Biology

Molecular Techniques Third-year Biology PLANNING Genetics Lab practices Molecular Techniques. Genetics Lab practices protocol. 2015-16 PCR-DIRECTED MUTAGENESIS, MOLECULAR CLONING AND RESTRICTION ANALYSIS Sessions 1 & 2 (2x3 hours): PCR-directed

More information

Cat. # Product Size DS130 DynaExpress TA PCR Cloning Kit (ptakn-2) 20 reactions Box 1 (-20 ) ptakn-2 Vector, linearized 20 µl (50 ng/µl) 1

Cat. # Product Size DS130 DynaExpress TA PCR Cloning Kit (ptakn-2) 20 reactions Box 1 (-20 ) ptakn-2 Vector, linearized 20 µl (50 ng/µl) 1 Product Name: Kit Component TA PCR Cloning Kit (ptakn-2) Cat. # Product Size DS130 TA PCR Cloning Kit (ptakn-2) 20 reactions Box 1 (-20 ) ptakn-2 Vector, linearized 20 µl (50 ng/µl) 1 2 Ligation Buffer

More information

Construction of a Mutant pbr322 Using Site-Directed Mutagenesis to Investigate the Exclusion Effects of pbr322 During Co-transformation with puc19

Construction of a Mutant pbr322 Using Site-Directed Mutagenesis to Investigate the Exclusion Effects of pbr322 During Co-transformation with puc19 Construction of a Mutant pbr322 Using Site-Directed Mutagenesis to Investigate the Exclusion Effects of pbr322 During Co-transformation with puc19 IVANA KOMLJENOVIC Department of Microbiology and Immunology,

More information

CLONING INVERTED REPEATS IN HIGH THROUGHPUT

CLONING INVERTED REPEATS IN HIGH THROUGHPUT CLONING INVERTED REPEATS IN HIGH THROUGHPUT This protocol is calculated for cloning one 96 well plate 1. design primers: as standard we include a EcoRI restriction site on the 5 primer and XbaI on the

More information

Cloning a Fluorescent Gene

Cloning a Fluorescent Gene Cloning a Fluorescent Gene Laboratory Protocols Handout v1.10 Table of Contents Lab 1: Pipettes and Pipetting... 2 Lab 2: Polymerase Chain Reaction... 5 Lab 3: Ligation... 7 Lab 4: Transformation... 9

More information

EZShuttle Recombination Cloning System

EZShuttle Recombination Cloning System EZRecombinase LR Mix Cat. No. RCBM-1001-020 (20 reactions) Cat. No. RCBM-1001-100 (100 reactions) EZRecombinase BP Mix Cat. No. ER003 (20 reactions) Cat. No. ER004 (100 reactions) User Manual GeneCopoeia,

More information

Table of contents. I. Flowchart of blunt end cloning of PCR products...2. II. Description...3. III. Kit Components...3

Table of contents. I. Flowchart of blunt end cloning of PCR products...2. II. Description...3. III. Kit Components...3 Table of contents I. Flowchart of blunt end cloning of PCR products...2 II. Description...3 III. Kit Components...3 IV. Reagents and Instruments Required...3 V. Storage...3 VI. About puc118 Hinc II/BAP...4

More information

HelixClone. PCR Cloning Kit. Simple and Fast technique for Cloning

HelixClone. PCR Cloning Kit. Simple and Fast technique for Cloning HelixClone PCR Cloning Kit Simple and Fast technique for Cloning www.nanohelix.net Contents Co ontents 1. Kit Contents 1) Product Types of PCR Cloning Kit 3 2) PCR Cloning Kit Reagents 3 3) Contents of

More information

igem2013 Microbiology BMB SDU

igem2013 Microbiology BMB SDU igem2013 Microbiology BMB SDU Project type: BioBrick Project title: DXS biobrick from B. subtilis#168 Sub project: Creation date: 12.07.13 Written by: Hwj Performed by: Hwj, MH, AK, PRA, SIS 1. SOPs in

More information

Transformation of DNA in competent E. coil

Transformation of DNA in competent E. coil Transformation of DNA in competent E. coil Reagents: SOC medium (1L) (a) 20g tryptone, 5g yeast extract, 0.5g NaCl in 950ml dh 2 O. (b) 250mM KCl: 1.86 KCl in 100ml dh 2 O. Add 10ml of solution (b) to

More information

YG1 Control. YG1 PstI XbaI. YG5 PstI XbaI Water Buffer DNA Enzyme1 Enzyme2

YG1 Control. YG1 PstI XbaI. YG5 PstI XbaI Water Buffer DNA Enzyme1 Enzyme2 9/9/03 Aim: Digestion and gel extraction of YG, YG3, YG5 and 8/C. Strain: E. coli DH5α Plasmid: Bba_J600, psbc3 4,, 3 6 7, 8 9 0, 3, 4 8/C 8/C SpeI PstI 3 3 x3 YG YG PstI XbaI.5 3.5 x YG3 YG3 PstI XbaI

More information

Mighty Cloning Reagent Set (Blunt End)

Mighty Cloning Reagent Set (Blunt End) Cat. # 6027 For Research Use Mighty Cloning Reagent Set (Blunt End) Product Manual Table of Contents I. Flowchart of blunt end cloning of PCR products...3 II. Description...4 III. Components...4 IV. Materials

More information

Gateway Vectors for BiFC

Gateway Vectors for BiFC Gateway Vectors for BiFC 1. The enhanced YFP (EYFP) are used (Split EYFP). 2. The Fusion fusion gene is expressed by CaMV35S promoter. 3. The N- or C-terminal fragments of EYFP are fused subsequent to

More information

Capsule deletion via a λ-red knockout system perturbs biofilm formation and fimbriae expression in Klebsiella pneumoniae MGH

Capsule deletion via a λ-red knockout system perturbs biofilm formation and fimbriae expression in Klebsiella pneumoniae MGH Capsule deletion via a λ-red knockout system perturbs biofilm formation and fimbriae expression in Klebsiella pneumoniae MGH 78578 Tzu-Wen Huang 1,2, Irene Lam 2, Hwan-You Chang 3, Shih-Feng Tsai 1, Bernhard

More information

Genlantis A division of Gene Therapy Systems, Inc Telesis Court San Diego, CA USA Telephone: or (US toll free)

Genlantis A division of Gene Therapy Systems, Inc Telesis Court San Diego, CA USA Telephone: or (US toll free) TurboCells BL21(DE3) TurboCells BL21(DE3)pLysS Chemically Competent E. coli Instruction Manual Catalog Numbers C302020 C303020 A division of Gene Therapy Systems, Inc. 10190 Telesis Court San Diego, CA

More information

ITS Sequencing in Millepora. 10/09 Subcloning DNA Fragments into pbluescript Preparation of pbluescript Vector

ITS Sequencing in Millepora. 10/09 Subcloning DNA Fragments into pbluescript Preparation of pbluescript Vector Page 1 of 5 10/09 Subcloning DNA Fragments into pbluescript Preparation of pbluescript Vector 1. Digest 1 µg of pbluescript with Eco RI 2. Following digestion, add 0.1 volumes of 3M sodium acetate (ph

More information

Mighty Cloning Reagent Set (Blunt End)

Mighty Cloning Reagent Set (Blunt End) Cat. # 6027 For Research Use Mighty Cloning Reagent Set (Blunt End) Product Manual Table of Contents I. Flowchart of blunt end cloning of PCR products...3 II. Description...4 III. Components...4 IV. Materials

More information

Linköpings Universitet. Site-directed mutagenesis of proteins

Linköpings Universitet. Site-directed mutagenesis of proteins IFM/Kemi August2011/LGM Linköpings Universitet Site-directed mutagenesis of proteins Competent E. coli cells Site-specific mutagenesis Analysis on agarose gel Transformation of plasmids in E. coli Preparation

More information

Presto Mini Plasmid Kit

Presto Mini Plasmid Kit Instruction Manual Ver. 03.06.17 For Research Use Only Presto Mini Plasmid Kit PDH004 (4 Preparation Sample Kit) PDH100 (100 Preparation Kit) PDH300 (300 Preparation Kit) Advantages Sample: 1-7 ml of cultured

More information

Supplementary Methods pcfd5 cloning protocol

Supplementary Methods pcfd5 cloning protocol Supplementary Methods cloning protocol vermilion trna grna trna grna U6:3 Terminator AmpR attb is a vector for expressing one or multiple trna-flanked Cas9 grnas under the control of the strong, ubiquitous

More information

Experiment (CSS451, 2010) 1. It is the. the amount. a. The ph of the. reaction solution, be stored at 4. VERY. -20oC. They. conditions.

Experiment (CSS451, 2010) 1. It is the. the amount. a. The ph of the. reaction solution, be stored at 4. VERY. -20oC. They. conditions. Experiment 3: Plasmid Digestion, Ligation, E coli. Transformation, and Selection (CSS451, 2010) GENERAL COMMENTS 1. It is the accepted convention that 1 unit of restriction endonuclease corresponds to

More information

Generation of Non-typeable Haemophilus influenzae Directed Gene Deletion Mutants Jeroen D. Langereis *

Generation of Non-typeable Haemophilus influenzae Directed Gene Deletion Mutants Jeroen D. Langereis * Generation of Non-typeable Haemophilus influenzae Directed Gene Deletion Mutants Jeroen D. Langereis * Laboratory of Pediatric Infectious Diseases, Department of Pediatrics and Laboratory of Medical Immunology,

More information

FROM EXPERIMENTS IN BACTERIAL GENETICS AND GENE TECHNIQUE

FROM EXPERIMENTS IN BACTERIAL GENETICS AND GENE TECHNIQUE Uppsala 2001-04-01 REPORT FROM EXPERIMENTS IN BACTERIAL GENETICS AND GENE TECHNIQUE Laboratory assistants: Maria Jönsson Amera Gibreel Students: Contents ASSIGNMENT:... 3 INTRODUCTION:... 3 MATERIAL AND

More information

Farnham Lab Protocol for CRISPR/Cas 9- mediated enhancer deletion using a puromycin selection vector Created by Yu (Phoebe) Guo

Farnham Lab Protocol for CRISPR/Cas 9- mediated enhancer deletion using a puromycin selection vector Created by Yu (Phoebe) Guo Farnham Lab Protocol for CRISPR/Cas 9- mediated enhancer deletion using a puromycin selection vector Created by Yu (Phoebe) Guo 20151024 Part A. Design grna oligos 1. Design grnas Go to Optimized CRISPR

More information

Electrocomp GeneHogs E. coli One Shot Electrocomp GeneHogs E. coli

Electrocomp GeneHogs E. coli One Shot Electrocomp GeneHogs E. coli Electrocomp GeneHogs E. coli One Shot Electrocomp GeneHogs E. coli Catalog nos. C8080-10, C8080-03, C800-05 Version E 17 May 2007 25-0387 www.invitrogen.com Overview Introduction The information in this

More information

VDL100.2 CLONING TRANSGENE INTO padenox

VDL100.2 CLONING TRANSGENE INTO padenox 1. Purpose 1.1. The purpose of this protocol is to transfer a transgene from the pshuttlex plasmid to padenox. 1.2. The starting material is 10 μg plasmid DNA. 1.3. This procedure is routinely performed

More information

For designing necessary primers and oligos for grna, online CRISPR designing tools can be used (e.g.

For designing necessary primers and oligos for grna, online CRISPR designing tools can be used (e.g. DESIGN OF grnas For designing necessary primers and oligos for grna, online CRISPR designing tools can be used (e.g. http://crispr.mit.edu/, https://benchling.com). Assembly of grna transcriptional cassettes

More information

igem2013 Microbiology BMB SDU

igem2013 Microbiology BMB SDU igem2013 Microbiology BMB SDU Project type: Biobrick Project title: DXS biobrick from B. subtilis Sub project: Creation date: 18.06.13 Written by: HWJ, PRA & SF Performed by: HWJ, ASF, SIS, MHK, SF & PRA

More information

Bethany and Elyse miniprepped the promoter, GFP and terminator cultures. Liz nanodropped the miniprep samples to obtain concentrations.

Bethany and Elyse miniprepped the promoter, GFP and terminator cultures. Liz nanodropped the miniprep samples to obtain concentrations. July 2: Liz and Bethany made glycerol stocks of the 12 FtsZ cultures, using 500 µl of 100% glycerol and 500 µl of the bacteria, and stored them in the -80ºC freezer. July 3: July 5: Bethany and Elyse miniprepped

More information

Gene Jockeying: Tricks of the Trade so that your Ligations work Every Time! Bevin Engelward

Gene Jockeying: Tricks of the Trade so that your Ligations work Every Time! Bevin Engelward Gene Jockeying: Tricks of the Trade so that your Ligations work Every Time! Bevin Engelward We will first review a simple example of a ligation. Know Your Vectors! -Find out as much as you can about your

More information

Procedure & Checklist - Preparing SMRTbell Libraries using PacBio Barcoded Universal Primers for Multiplex SMRT Sequencing

Procedure & Checklist - Preparing SMRTbell Libraries using PacBio Barcoded Universal Primers for Multiplex SMRT Sequencing Procedure & Checklist - Preparing SMRTbell Libraries using PacBio Barcoded Universal Primers for Multiplex SMRT Sequencing Before You Begin This document describes methods for generating barcoded PCR products

More information

Title: Understanding the impact of orientation on gene expression of lux operon in pkn800 transformation into Escherichia coli DH5α

Title: Understanding the impact of orientation on gene expression of lux operon in pkn800 transformation into Escherichia coli DH5α Seim - 1 Name: Darian Seim Title: Understanding the impact of orientation on gene expression of lux operon in pkn800 transformation into Escherichia coli DH5α Date: April 12 th, 2016 April 18 th, 2016

More information

Site-directed mutagenesis of proteins

Site-directed mutagenesis of proteins IFM/Kemi Linköpings Universitet August 2013/LGM Labmanual Site-directed mutagenesis of proteins Figur 1: Flow-chart of the site-directed mutagenesis lab exercise 2 Site-specific mutagenesis Introduction

More information

Engineering D66N mutant using quick change site directed mutagenesis. Harkewal Singh 09/01/2010

Engineering D66N mutant using quick change site directed mutagenesis. Harkewal Singh 09/01/2010 Engineering D66N mutant using quick change site directed mutagenesis Harkewal Singh 09/01/2010 1 1- What is quick change site directed mutagenesis? 2- An overview of the kit contents. 3- A brief information

More information

Protocols for cell lines using CRISPR/CAS

Protocols for cell lines using CRISPR/CAS Protocols for cell lines using CRISPR/CAS Procedure overview Map Preparation of CRISPR/CAS plasmids Expression vectors for guide RNA (U6-gRNA) and Cas9 gene (CMV-p-Cas9) are ampicillin-resist ant and stable

More information

Generation of gene knockout vectors for Dictyostelium discoideum

Generation of gene knockout vectors for Dictyostelium discoideum Generation of gene knockout vectors for Dictyostelium discoideum Instruction manual Last date of revision April 2012 2015 Version PR29-0001 PR29-0003 www.stargate.com This manual can be downloaded under

More information

Guide-it Indel Identification Kit User Manual

Guide-it Indel Identification Kit User Manual Clontech Laboratories, Inc. Guide-it Indel Identification Kit User Manual Cat. No. 631444 (120114) Clontech Laboratories, Inc. A Takara Bio Company 1290 Terra Bella Avenue, Mountain View, CA 94043, USA

More information

Restriction Analysis of Purified para-r

Restriction Analysis of Purified para-r Restriction Analysis of Purified para-r INTRODUCTION The restriction analysis will provide final proof that the cells transformed during Laboratory 6, cloned overnight in LB/amp and purified in Lab 10

More information

PrecisionX Multiplex grna Cloning Kit. Cat. # CAS9-GRNA-KIT. User Manual

PrecisionX Multiplex grna Cloning Kit. Cat. # CAS9-GRNA-KIT. User Manual PrecisionX Multiplex grna Cloning Kit Store at -20 C upon receipt A limited-use label license covers this product. By use of this product, you accept the terms and conditions outlined in the Licensing

More information

General Molecular cloning Protocols (Subcloning a 300bp fragment into a 5kp vector) Design Primers. PCR Reaction. DNA Electrophoresis

General Molecular cloning Protocols (Subcloning a 300bp fragment into a 5kp vector) Design Primers. PCR Reaction. DNA Electrophoresis General Molecular cloning Protocols (Subcloning a 300bp fragment into a 5kp vector) Design Primers 1. 18-25 bp overlapping with the desired PCR fragment, with 5-6 extra base pairs and the DNA restriction

More information

Puro. Knockout Detection (KOD) Kit

Puro. Knockout Detection (KOD) Kit Puro Knockout Detection (KOD) Kit Cat. No. CC-03 18 Oct. 2016 Contents I. Kit Contents and Storage II. Product Overview III. Methods Experimental Outline Genomic DNA Preparation Obtain Hybrid DNA Digest

More information

Made 1,2 % agarose gel with ETBR. Ran 5 ul samples of each PCR reaction with 1 ul LD on gel: 20 min, 120V. Used Gene O'Ruler 1 kb ladder.

Made 1,2 % agarose gel with ETBR. Ran 5 ul samples of each PCR reaction with 1 ul LD on gel: 20 min, 120V. Used Gene O'Ruler 1 kb ladder. 10.8.2015 MONDAY, 8/10 Petra, Tamannae Did a new PCR reaction for amphiphilic protein with linker, because purification of the reaction done last week was unsuccesful (A260/A280: 1,12). Chose Tm according

More information

Supplementary Figure 1 Gel electrophoresis of Ligation assembled GFP expression circuits.

Supplementary Figure 1 Gel electrophoresis of Ligation assembled GFP expression circuits. Supplementary Figure 1 Gel electrophoresis of Ligation assembled GFP expression circuits. Verification was performed by digestion with restriction enzymes EcoRI and PstI, resulting in 4,413 kb and 949

More information

0730 Measure the concentrations of Ligation products of reverse laci + promoter

0730 Measure the concentrations of Ligation products of reverse laci + promoter 0730 Measure the concentrations of Ligation products of reverse laci + promoter 1 1.6536 2 2.4225 3 2.7404 4 2.8690 5 2.4858 6 2.5618 Enzyme digestion to test the ligation product Plasmid 8µl EcoRI 1.5

More information

EZ-10 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK

EZ-10 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK EZ-0 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK (Bacteria, Plant, Animal, Blood) Version 8 Rev 05/0/03 EZ-0 Genomic DNA Kit Handbook Table of Contents Introduction Limitations of Use Features Applications

More information

Petra, Tamannae. Made new 1:10 dilutions of P001 and P ul primer to 18 ul water

Petra, Tamannae. Made new 1:10 dilutions of P001 and P ul primer to 18 ul water 20.7.2015 MONDAY, 7/20 Petra, Tamannae Made new 1:10 dilutions of P001 and P015. 2 ul primer to 18 ul water Made new 1 ng/µl template dilution of CAR part 1. 2 µl DNA stock and 18 µl H2O Gradient PCR for

More information

User s Guide Catalog number (8 reactions), (16 reactions), and (24 reactions)

User s Guide Catalog number (8 reactions), (16 reactions), and (24 reactions) GenEdit Site-Directed DNA Mutagenesis Kit For Medium and Large Plasmids User s Guide Catalog number 201321 (8 reactions), 201322 (16 reactions), and 201323 (24 reactions) For Research Use Only Not for

More information

BRED: Bacteriophage Recombineering with Electroporated DNA

BRED: Bacteriophage Recombineering with Electroporated DNA BRED: Bacteriophage Recombineering with Electroporated DNA Introduction We have developed a system for generating mutations in lytically replicating mycobacteriophages that we have termed BRED: Bacteriophage

More information

Vector Linearization. igem TU/e 2016 Biomedical Engineering

Vector Linearization. igem TU/e 2016 Biomedical Engineering igem TU/e 2016 Biomedical Engineering Eindhoven University of Technology Room: Ceres 0.04 Den Dolech 2, 5612 AZ Eindhoven The Netherlands Tel. no. +31 50 247 55 59 2016.igem.org/Team:TU_Eindhoven Vector

More information

Vector Linearization. igem TU/e 2015 Biomedical Engineering

Vector Linearization. igem TU/e 2015 Biomedical Engineering igem TU/e 2015 Biomedical Engineering Eindhoven University of Technology Room: Ceres 0.04 Den Dolech 2, 5612 AZ Eindhoven The Netherlands Tel. no. +31 50 247 55 59 2015.igem.org/Team:TU_Eindhoven Vector

More information

Enhanced Arginase production: rocf

Enhanced Arginase production: rocf Enhanced Arginase production: rocf Purpose and Justification: Bacillus subtilis produces urease, which catalyses the hydrolysis of urea into ammonium and carbonate. Since the cell wall of the bacteria

More information

EXPERIMENT 4 CLONING THE UPSTREAM REGION OF THE GENE OF INTEREST

EXPERIMENT 4 CLONING THE UPSTREAM REGION OF THE GENE OF INTEREST EXPERIMENT 4 CLONING THE UPSTREAM REGION OF THE GENE OF INTEREST Purpose: To determine the activity of the promoter of the gene of interest at the cellular and tissue levels in Arabidopsis plants via the

More information

DNA Ligation Kit Ver. 1 Manual

DNA Ligation Kit Ver. 1 Manual Table of content Description... 2 Procedures and Examples A. Insertion of DNA into plasmid vectors... 3 B. Insertion of DNA into λ phage vectors... 4 C. Self-circulization of linear DNA... 4 D. Linker

More information

Bio 121 LAB 11 INSTRUCTIONS - DNA II

Bio 121 LAB 11 INSTRUCTIONS - DNA II Bio 121 LAB 11 INSTRUCTIONS - DNA II In the first part of today's lab we will demonstrate that the DNA which we extracted last week can create heritable changes in the phenotype of bacterial cells. We

More information

Cloning of pooled sgrnas into lentiviral vector Developed by JEV, LAG and MH Modified and updated, RAP & CYP

Cloning of pooled sgrnas into lentiviral vector Developed by JEV, LAG and MH Modified and updated, RAP & CYP Cloning of pooled sgrnas into lentiviral vector Developed by JEV, LAG and MH Modified and updated, RAP & CYP 1) Vector Preparation: plg1 library vector a) Digest 5 ug of vector with Thermo Scientific FastDigest

More information

GeNei TM Transformation Teaching Kit Manual

GeNei TM Transformation Teaching Kit Manual Teaching Kit Manual Cat No. New Cat No. KT07 107385 KT07A 106220 Revision No.: 00060505 CONTENTS Page No. Objective 3 Principle 3 Kit Description 6 Materials Provided 7 Procedure 9 Observation & Interpretation

More information

Complete protocol in 110 minutes Enzymatic fragmentation without sonication One-step fragmentation/tagging to save time

Complete protocol in 110 minutes Enzymatic fragmentation without sonication One-step fragmentation/tagging to save time Molecular Cloning Laboratories Manual Version 1.2 Product name: MCNext UT DNA Sample Prep Kit Cat #: MCUDS-4, MCUDS-24, MCUDS-96 Description: This protocol explains how to prepare up to 96 pooled indexed

More information

Basic Protocol (v. 2.0, May, 2003)

Basic Protocol (v. 2.0, May, 2003) Basic Protocol (v. 2.0, May, 2003) Preparation of RNA:DNA Handles For the two handles (called A and B), you will need the following oligos: Product 1 : Name=B_reverse : Synthesis=1 umole : Purification=HPLC

More information