Dr Sameera Hassan Assistant Professor 15 th October

Dr. Sameera Hassan Assistant Professor 15 th October, 2019

• PCR is a means to amplify a particular piece of DNA • Amplify= making numerous copies of a segment of DNA • PCR can make billions of copies of a target sequence of DNA in a few hours • PCR was invented in the 1984 as a way to make numerous copies of DNA fragments in the laboratory • Its applications are vast and PCR is now an integral part of Molecular Biology

Buffer Mg. Cl 2 d. NTPs Primers Template Taq DNA Polymerase Water

TAQ POLYMERASE • catalyzes the elongation of DNA by adding nucleoside triphosphates to the 3’ end of the growing strand • A nucleotide triphosphate is a 1 sugar + 1 base + 3 phosphates • When a nucleoside triphosphate joins the DNA strand, two phosphates are removed. • DNA polymerase can only add nucleotides to 3’ end of growing strand • It adds A overhangs as an its inherent property which is the basis of TA cloning

q The specificity of primer to template hybridization will depend upon temperature and salt q The highest annealing temperature possible should be used to reduce nonspecific associations. q Some nonspecific amplifications can be avoided by employing a “hot-start” technique—i. e. , adding the DNA polymerase to a prewarmed sample q purifying the PCR product by gel electrophoresis will help ensure that the proper DNA fragment is subcloned. q for cloning, fidelity is more important than yield, so it is better to keep a low cycle number and q not to raise the Mg. Cl 2 concentra- tion too much. 1. 5 m. M Mg. Cl 2 in the amplification buffer should be sufficient for most primers. q Vent polymerase q Taq DNA polymerase lacks the 3′→ 5′ proofreading exonuclease activity and results in a heightened error frequency. 0. 02% per position q Pfu DNA polymerase, purified from Pyrococcus furiosus (Stratagene) and V ent DNA polymerase, purified from Thermococcus litoralis q Pfu DNA polymerase is 12 -fold more accurate than Taq DNA polymerase q V ent DNA polymerase is 4 -fold more accurate than Taq DNA polymerase

TA CLONING

TA CLONING Ligation Transformation Miniprep Restriction Digestion

PLASMID ISOLATION Plasmids Electron micrograph of DNA from a lysed E. coli cell

MOLECULAR BIOLOGY APPLICATIONS FOR PLASMIDS 2. Sequencing 3. Transfection 4. Gene therapy

Supercoiled Open Circular Linear, nicked

AGAROSE GEL ELECTROPHORES IS

Electrophoresis solution containing either a weak acid and its salt or a weak base and its salt Agarose Method that uses an electrical current and a gel matrix to separate molecules. carbohydrate AGAROSE GEL ELECTROPHORESIS Buffer

Agarose is a polysaccharide (carbohydrate) polymer material, generally extracted from seaweed Alternating sugar units form linear chains Cross-linking chains via hydrogen forms porous matrix Heat breaks matrix apart Cooling forms a matrix with an “average” pore size

• Ethidium Bromide a fluorescent chemical that intercalates between base pairs in a double stranded DNA molecule. Commonly used to detect DNA following gel electrophoresis. • DNA Ladder consists of known DNA sizes used to determine the size of an unknown DNA sample. The DNA ladder usually contains regularly spaced sized samples which when run on an agarose gel looks like a "ladder". • Power Supply a source of electric current

Buffers The most common being: Ø Tris/Acetate/EDTA (TAE) Ø Tris/Borate/EDTA (TBE) Ø Ethidium bromide stock in the gel solution for a final concentration of 0. 5 ug/ml.

Gel Percentage Optimal Resolution Range 0. 75% 500 bp– 10 kb 1. 00% 300 bp– 9 kb 1. 25% 100 bp– 8 kb Gel Percentage Optimal Resolution Range 2% 100 bp– 2. 5 kb 3% 40 bp– 2 kb 4% 20 bp– 1 kb

Ethidium bromide Syber Green Crystal Violet Methylene Blue