Post midsem

:
Recombinant DNA technology for protein production

1) Plasmid isolation using alkaline lysis method

2) Restriction digestion and Agarose gel electrophoresis

3) Transformation of Plasmid DNA

4) Protein isolation and SDS-PAGE

Restriction Digestion of Plasmid
DNA
Jeet Kalia

Basic structure of a Cloning and Expression Plasmid Vector

promoter

transcription
termination
Multiple cloning site sequence
(MCS)

Selection
marker
origin of
replication

Basic structure of a Cloning and Expression Plasmid Vector • MCS: This is a synthetic stretch of DNA and accomodates a string of unique restriction sites. Kanamycin. Tetracycline etc) and helps in selection of positive transformants • Promoter: This generally is a viral promoter and is where the RNA polymerase binds to drive the transcription of downstream foreign gene • Terminator sequence: This transcribes a stop codon downstream of the MCS .r. It facilitates cloning of a foreign DNA in an ORF w.t the promoter • Origin of replication (ori): This is a replicon of bacterial or phage origin and ensures replication of plasmid DNA • Selection marker: This generally is a gene coding for an antibiotic resistance( Ampicillin.

Pfu pET-17b: Plasmid Map 21-03-2017 BIO122 5 Source: Novagen .

Restriction enzymes • Restriction enzymes/ restriction endonucleases (RE) : – a special class of endonucleases found in bacteria – digest DNA by recognizing specific short sequences of 4. 6 or 8 base pairs – Palindromic sequences • Palindromic sequence : that reads the same from both sides • Examples: 5’-GAATTC-3’ 3’-CTTAAG-5’ • Cleaves the phosphodiester bond of the DNA backbone .

What are restriction enzymes? Evolved by bacteria to protect against viral DNA infection – 1950s: discovery of primitive immune system in bacteria – Werner Arber and Matthew Meselson got the nobel prize in 1960 for first identifying these enzymes in E.139 known enzymes .adds methyl group to its bases Endonucleases = cleave within DNA strands Exonucleases = digest from the ends of DNA molecules Types I. – Host DNA is marked up by Methylase enzyme. III and IV 3. II. coli.

Types of RE • Traditionally classified into four types on the basis of :- – subunit composition – cleavage position – sequence specificity – cofactor requirements. .

and Mg2+ ions. but they have little practical value since they do not produce discrete restriction fragments or distinct gel-banding patterns. hydrolyzed adenosine triphosphate (ATP). • Cofactors: S-Adenosyl methionine (Ado-met). • Of considerable biochemical interest. Type I RE’s • Complex. multisubunit and heterodimer. . combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences.

• Subunits are in the range of 250-300 amino acids • Mostly recognize symmetric DNA sequences as they bind as homodimers • The only class used in the laboratory for routine DNA analysis and gene cloning since they produce discrete restriction fragments and distinct gel banding patterns. Type II RE’s • Homodimer. cleaves DNA near or within the recognition sites. . only requires Mg2+ ions as cofactors. • Do not use ATP. recognition sites are mostly palindromic (4-8 bp).

subsequent ligation is very specific Enzyme cuts  5’ GAATTC 3’ 3’ CTTAAG 5’ 5’ G 3’ 3’ CTTAA 5’ 5’ AATTC 3’ 3’ G 5’ Generates 5’ overhang (in the 5’ direction) Blunt ends: subsequent ligation is non. 5’ GAA TTC 3’ specific 3’ CTT AAG 5’ . Palindromic Sequences 5’ versus 3’ overhang: Sticky Ends.

Molecular cloning • Overhangs or sticky ends • Blunt ends .

• Contains more than one subunit • Requires ATP Type IV RE’s • Recognizes modified. typically methylated DNA.Type III RE’s • recognize two separate non-palindromic sequences that are inversely oriented. • Cleave 20-30bp outside recognition sequences. .

coli R: I: first strain enzyme of that type isolated BamHI Bam: Bacillus H: I: first amyloliquefaciens strain enzyme of that type isolated Sau3A Sau: Staphylococcus 3A: aureas strain . EcoRI Eco: E. How are they named • Restriction enzymes are named by the organism from which they were first isolated.

What happens if temperature is too hot or cool? . Restriction Enzyme Digestion An Enzymatic Unit (u) is defined as the amount of enzyme required to digest 1 ug of DNA under optimal conditions in 1 hour. Restriction Buffer provides optimal conditions: NaCl provides correct ionic strength Tris-HCl provides the proper pH Mg2+ is an enzyme co-factor Why incubate at 370C? Body temperature is optimal for these and most other enzymes.

Electrophoresis: a method of separating charged molecules in an electrical field. it will move towards the positive end (cathode to anode). Size and Conformation (shape) . The three-dimensional matrix of the agarose gel is brought about by non-covalent bonds formed between polysaccharide units. DNA has an overall negative charge. How do we visualize the DNA? Agarose gel electrophoresis Agarose : polysaccharide extracted from the cell wall of red algae.

How do we visualize the DNA? .

Cathode - Anode + Buffer Dyes Agarose gel Power Supply .

Establish pH and provide ions to support conductivity. Contains a dense substance. such as glycerol. Loading dye:DNA samples are loaded into a gel AFTER the tank has been filled with buffer. covering the gel. DNA staining: Allow DNA visualization. Ethidium bromide or Sybrsafe stains . to allow the sample to "fall" in the well Contains one or two tracking dyes. which migrate in the gel and allow monitoring of how far the electrophoresis has proceeded. Components of an Electrophoresis System Electrophoresis Buffer:TAE (Tris-acetate-EDTA) and TBE (Tris- borate-EDTA) are the most common buffers for duplex DNA.

Running plasmid DNA on an agarose gel http://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/ .

Restriction Digestion Analysis .

. close the tube and centrifuge for a few seconds. Protocol for restriction digestion Component Volume Sample Plasmid (DNA) 3 μl Restriction Enzyme 10X Buffer 2 μl Restriction Enzyme EcoRI 0. Incubate at the 37 deg C (enzyme’s optimum temperature) for 45 min.5 μl Restriction Enzyme HindIII 0.5 μl Water 14 μl Final volume 20μl Mix gently.

Load the sample on 1% agarose gel and run it at 100 volts. . Add loading buffer to a 1X final concentration and proceed to gel electrophoresis. Protocol for Agarose Electrophoresis 1. 3. Examine the gel under the transilluminator once the dye front traverses about 2/3 of the gel's length. Capture the gel picture. 2.

Questions: Restriction mapping .

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