Protocols

Display Protocols
<< previous | next >>
AbbreviationNameLabTypeDescription
Cloning_TorontoCloning_TorontoUTcloning1. PCR Amplification /Rxn dH2O 34.1 µl 10x Pfx buffer 5.0 µl 50 mM MgSO4 1.0 µl 10 mM dNTP’s 1.5 µl 10x Enhancer 7.5 µl genomic ...
His-Trap purificationThis is a purification using the HisTrap column of theUVApurificationBinding buffer: 50 mM Sodium Phosphate pH 8 500 mM NaCl 10 mM Imidazole Elution Buffer: 50 mM Sodium Phosphate pH 8 500 mM NaCl 250 mM Imidazole Crystalliztion Buffer 20 mM HEPES pH 7 1 ...
NU_tag-off_purificationNU tag-off purificationNUpurificationProteins are purified by Ni-affinity chromatography on HiTrep FF and desalting HiPrep 20/10 columns using AKTA Express system. Pure proteins are collected in buffer, containing 10 mM Tris-HCl (pH 8.3, ...
PKS13 PurificationGeneral protocol described by Dr. Courtney Aldrich in order to express and purify PKS13 target. The target was sent to NU with the following letter and descriptions.NUpurificationSeptember 16, 2015 Dr. Elisabetta Sabini Northwestern University Feinberg School of Medicine Morton #7-605 303 E. Chicago Ave. Chicago, IL 60611 Phone (312) 503-6973 Email: e-sabini@northwest ...
SmallScaleExpression_protocol_TorontoSmallScaleExpression_protocol_TorontoUTexpressionSmall scale expression DAY 1 1.Place PCR plate containing the plasmids (3 µl at 50-100ng/ul) on ice ~10 min. 2.Add 25-30 µl of competent BL21-CodonPlus(DE3)-RIL cells (Stratagene). 3.Leave on ...
standard cloning_ANLstandard cloning_ANLANLcloningDay 1: Setup PCR plate, and run it in the thermocycler overnight. This protocol will produce a well volume of 51 µL: 0.26 mM each primer 1x buffer 0.2 mM each dNTP 1 mM MgSO4 0.016 U/ ...
standard crystallization_ANLstandard crystallization_ANLANLcrystallizationCRYSTALLIZATION SETUP 1 Concentrate protein samples using Centricon Concentrators 5K. From this point on the work is conducted with concentrated proteins and extra care should be taken. The Centri ...
standard expression_ANLstandard expression_ANLANLexpressionOverview: Grow cultures, induce protein expression. Materials: M9 components. Deepwell plates Thermocycler plates Notes: The red solution includes 100 mg/mL amp. Day 1: Starter Cu ...
standard purification_ANLstandard purification_ANLANLpurificationDay 1 – Small Culture (50mL) Pick up cell culture from cloning lab. They will be in small, 2mL test tubes. Each tube contains a different target protein cloned in pMCSG7 (or other MCSG) vector an ...
UTSW_Purification_HPLCusing FPLCUTSWpurificationFPLC Protocol Autoclave LB medium and 50ml flasks Inoculate glycerol stock in 50ml of LB medium + Antibiotic O/N @ 37°C Amp – 50µl of 100mg/ml [final conc. 200µg/ml] Kan - 50µl of 100µg/m ...
UTSW_standard_cloningUTSW_standard_cloningUTSWcloningCloning Transformation Protocol: Transformation of clone into 2 competent cells 1) One shot Top 10 competent cells 2) Rosetta (DE3) pLysS competent cells Thaw the competent cells (50ml) on ...
UTSW_standard expressionUTSW_standard expressionUTSWexpressionCloning Transformation Protocol: Transformation of clone into 2 competent cells 1) One shot Top 10 competent cells 2) Rosetta (DE3) pLysS competent cells Thaw the competent cells (50ml) on ...
UTSW_standard purificationUTSW_standard purificationUTSWpurificationPurification Cell disruption: 1. Spin 1L bacterial cultures at 4000rpm 40mins at 4 ºC. 2. Prepare BB buffer. 3. Decant the media (incubate with bleach for at least 1hour before you pour it into ...
UVA_default_crystallizationUVA_default_crystallizationUVAcrystallizationStandard Virginia Crystalliation protcol: 1. After purification concentrate your sample to conc. 5-20 mg/ml using Amicon concentrators with proper MW membrane cut-off. It's better to prepare more ...
uva_expression_native_1typical native expression protocol at Wladek Minor groupUVAexpressionExpression of Native APC Proteins Preparation (within 1-3 days prior) 1)Transform the vectors of interest into the BL21(DE3)-derived expression strain OR plate frozen expression stocks on fresh pl ...
uva_expression_semet_1typical semet expression protocol at Wladek Minor groupUVAexpressionExpression of Seleno-Methionine-Labelled APC Proteins The general procedure is to grow a small seed culture (10mL) of a methionine auxotrophic strain in M9 minimal media plus Met. This overnight cult ...
uva_purification_1typical purification protocol at Wladek Minor groupUVApurificationProtein Purification Protocol BUFFERS All of these buffers contain 5mM beta-mercaptoethanol(BME) if proteins highly susceptible to oxidation such as Se-Met proteins or proteins with high cystein ...
uva_purification_refoldingProtocol for purification in denaturing conditionsUVApurification1.Resuspend insoluble pellet in BB8MU ~30 min, RT, vortex 2. You can sonicate the pellet if it is still viscous 3.Spin down the lysate 35 000 rpm 4C 30 min – 1h collect samples of soluble and ...
WU_baculovirus_expression_1WU_baculovirus_expression_1WUselectionRecombinant baculovirus were generated with flashBAC baculovirus expression system (Mirus). Basically, the ectodomain of HA was cloned into a modified pOET1 vector (see cloning method) and co-transfec ...
WU_basic_cloning_protocol_1WU_basic_cloning_protocol_1WUcloningDay 1: Setup PCR. Prepare 100 µM stock solutions of the forward and reverse PCR primers in TE buffer. Prepare template DNA stock at a concentration of 10 ng/µl in TE buffer. For a 50 µl PCR reacti ...
WU_crystallization_protocol_1WU_crystallization_protocol_1WUcrystallizationDefault Washington University crystallization setup: Purified proteins are exchanged into crystallization buffer (20 mM HEPES pH 7.4, 25 mM NaCl) using a separate Amicon Ultra centrifugal filtration ...
WU_inclusion_body_refoldingWU_inclusion_body_refoldingWUpurificationInclusion body pellet is repeatedly washed in buffer containing : 50 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 0.01% sodium azide, 1 mM DTT, 100 mM sodium chloride, and 0.5% Triton X-100. Final wash i ...
WU_native_inclusion_body_expression_2WU_native_inclusion_body_expression_2WUpurificationExpression of natively-folded/soluble proteins in E. coli. Day 1. Streak an LB-carbenicillin plate from frozen stocks to obtain isolated colonies of the expression plasmid in E. coli strain BL21-Codo ...
WU_native_soluble_expression_1WU_native_soluble_expression_1WUexpressionExpression of natively-folded/soluble proteins in E. coli. Day 1. Streak an LB-carbenicillin plate from frozen stocks to obtain isolated colonies of the expression plasmid in E. coli strain BL21-Codo ...
WU_PEI_transient_transfectionWU_PEI_transient_transfectionWUexpressionLarge-scale Transient Transfection of Mammalian Cells in Suspension Culture. The majority of targets produced by the CSGID are expressed in E. coli as soluble-natively-folded proteins. This works wel ...
WU_SeMet_feedback_inclusion_body_2 WU_SeMet_feedback_inclusion_body_2 WUexpressionThe protein is expressed in E. coli strain BL21-CodonPlus (DE3)-RIL cells (Agilent Technologies Cat. No. 230245). There is no need for a methionine synthesis deficient strain. Feedback inhibition of ...
WU_SeMet_feedback_inhibition_soluble_1 WU_SeMet_feedback_inhibition_soluble_1 WUexpressionThe protein is expressed in E. coli strain BL21-CodonPlus (DE3)-RIL cells (Agilent Technologies Cat. No. 230245). There is no need for a methionine synthesis deficient strain. Feedback inhibition of ...
<< previous | next >>