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RF1- CaCl2, MgCl2, KCl	Ions used for creating competent cells, Ca2+ neutralises negative charge on membrane, Cl- enters cell to expand membrane and create holes.
RF2- Mops, KCl, CaCl2, glycerol	Mops- buffer pH 6.8 biological buffer KCl, CaCl2- already competent from RF1, just keeps them competent, glycerol lowers freezing temperature, prevents icicles.
LB	מצע עשיר, מורכב tryptone, yeast extract, NaCl, agar (for solid LB).
Competent E. coli	Need to be in late logarithmic stage of growth to create competent E. coli. If you measure OD at this stage at 600 nm, the reading should be 0.8
Tryptone (LB)	Originates from casein, provides amino acids and nitrogen
Yeast extract (LB)	Content of yeast cell without membrane, provides vitamins (B family, and K), sugars, minerals, small peptides
NaCl (LB)	Maintains osmotic pressure between bacteria cell and surroundings.
Agar (LB)	extracted from seaweed, causes solid texture for LB.
Ampicillin	from the penicillin family of antibiotics, includes a β- lactam ring which prevents formation of peptide bonds in peptidoglycan layer in bacteria.
IPTG	removes repressor lacZ gene operon
X-Gal	Broken down by β- galactosidase into galacltose + another molecule which turns blue when disconnected from galactose.
Transformation efficiency	Vitality of competent E. coli Supercoiled plasmid (supercoiled > linear > relaxed) 1:10,000 (expected success rate)
Cloning vectors	ORI recognised host polymerase Defined host Vector size- effects copy number small vector = large copy number & reverse Multiple Cloning site (targets for restriction enzymes) Selective marker (antibiotic resistance) reporter gene for ligation
pBluescript	phagemid lacZ - reporter gene- encodes to β- galactosidase MCS- on lacZ gene 21 restrictive enzyme sites amp resistance- selective marker Bla amp- β- lactamase
Plasmid extraction by alkaline extraction method	Cell resuspension solution (CRS)- TRIS, HCl, EDTA (removes metal cofactors of enzymes), RNase Cell lysis solution- basic pH, NaOH, and SDS Alkaline protease solution- protease which break down nuclease Neutralisation Solution (NSB)- guanidine hydrochloride (denaturation of proteins (nucleases) by breaking hydrogen bonds, and hydrophobic bonds, enhances binding of DNA to silica), K-Acetate
Restriction enzymes used to cut plasmid	BamHI- sticky ends 5' GATC PSTI- sticky ends TGCA 3'
DH5-	Type of E. coli developed for lab use mutations: lacZ mutation- allows for lacZ expression through plasmid vector and blue white screening. rec-A1: doesn't allow recombination of inserted vectors end- A1: low endonuclease action, allows for successful transformation
Electrophoresis buffer	TAEX1: Tris- contains ions which transfer electric pulse Acetic acid- buffer EDTA- neutralises enzymatic activity
Alkaline Phosphatase (AP)	At the end of the restriction enzyme process, AP is added to remove the phosphate group at the 5' end of the DNA strand in order to prevent the plasmid from re-closing.
Ethidium Bromide	aromatic molecule, attaches between the strands of DNA (which makes it cancerous). Absorbs ultra violet light, is used to measure outcome of electrophoresis gel.
Loading Dye and glycerol	includes 2 colors, Bromo-phenol blue (BPB) xylene cyanol. used to access how fast the gel is running glycerol- high density, DNA example sinks in the well and doesn't float and escape the well.
DNA (plasmid) purification	Binding Buffer- includes high salt concentration, pH<7.5 Isopopropanol Alcohol- DNA is insoluble in this material, allows for DNA pellet to form during centrifugation. Elution Buffer- low salt concentration, basic pH from Tris Cl 8.5
PCR- Materials	Two primers forward and reverse Taq Polymerase- thermophilic polymerase dNTPs template- DNA strand to be amplified Mg2+- cofactor of the polymerase
PCR- process	1. denaturation in high temp 95ºC 2. annealing of primers 65ºC 3. extension about 70ºC- Taq polymerase synthesises new cycles *replication is exponential (2^n)
Insert	mFer Tyrosine kinase- codes for the enzyme tyrosine kinase, involved in phosphorylation of proteins. 2500 bp
Restriction enzymes used to cut insert and pair to sticky ends of plasmid	Bgl2- 3' GATC PSTI - TGCA 3'
Primer design	Over 18 nucleotides Edge of primers must have MCS (MCS cannot include same sequence in the gene itself) Upstream to the primer there must be a tail of about 5 nucleotides to allow enzyme attachment Similar Tms (denaturation temperature) for both primers
Primer design cont.	GC % between 40-60%, too high, will bind to strongly, too low, bing weakly.
P	Activity Buffer w/ Taq polymerase and cofactor Mg2+ dNTPs
nanodrop	nucleic acids absorb light wave length 260 nm Salts 230 nm Proteins 280 nm purification from proteins 260/280 = 1.8 purification from salts 260/230 = 2 Measures sample volume of 1 µl
DNA ligase	creates phospho-di-ester bonds between 3'-OH and 5'- phosphate T4 DNA ligase- can join blunt ends or sticky ends of single or double strands. Can be inactivated at 70ºC. Requires ATP, pH 7.2-7.8
Ligase mechanism	Ligase is covalently bound to AMP, AMP is transferred to 5'- phosphate of the cut DNA stand, nucleophile attack by the 3'-OH on the AMP, creates a di-ester bond
Optimal conditions for succesfull ligation	1. Ration of insert to vector--> 3:1 2. Amount of DNA 10-200 ng of plasmid 3. volume of reaction 10 µl minimum 4. Amount of ligase, 1-10 units of ligase for each reaction
Potential problems with ligation	deterioration of buffer components- ATP... Restriction enzyme still active in ligation mixture Non specific nuclease contamination
Antibody נוגדן	Fab site- section 110-130 amino acids unique to each antigen, binds the antigen FC region- 5 different kinds, each one induces different immune reaction.
Conditions of Elisa	Antigen-Antibody bond Enzyme + specific substrate which form chromogenic reaction
Pros of Elisa method	qualitative and quantitative measurements visual outcome which can be measured quantitively by a spectrophotometer
Uses for Elisa method	Uses: steroid hormones, antibodies for bacteria and viruses, drugs, allergens
Non- competitive Antibody test (sandwich)	antigen specific to antibody that we want to check, connected to well, antibody which added, binds to antigen, secondary antibody w/ enzyme binds to first antibody, add substate, measure outcome. Outcome vs. calibration w/o target antibody
Non- competitive antigen test (sandwich)	Antibody with Fab region specific to target antigen is bound to well, target antigen is added, enzyme labeled antibody with identical Fab region is added, substrate added, measure outcome. Outcome vs. calibration w/o target antigen
Competitive test for antibody	Antigen specific to target antibody is added to well, two identical antibodies are added, one from the patient, other one w/ enzyme. Qualtiative outcome compared to antigen with enzyme antibody
Competitive test for antigen	Antigen identical to target antigen is added to well in a known concentration, target antigen with matching enzyme + antibody are added to well, target antigen competes with first antigen for bonds w/ antibody. Qualitative outcome compared to test w/o patient sample

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	Erstellt von Sarah Winkler vor mehr als 8 Jahre