PKS Plasmid : 20 ul 10XTango Buffer : 3 ul SmaI (1u/ul) : 1 ul Distilled dH2O : 6 ul 30 ul

Retinal cDNA Library

Blue-White Screening & α-complementation

* lacZ encodes β-galactosidase (LacZ), cleaves lactose into glucose and galactose. * lacY encodes β-galactoside permease (LacY) * lacA encodes β-galactoside transacetylase (LacA) lac Operon

β-galactosidase (LacZ), cleaves lactose into glucose and galactose.

lac operon inducer: the metabolite of lactose; allolactose Inducer: IPTG (Analog of allolactose) X-Gal (lactose an.)  Indigo Blue + galactose α-complementation

E. coli: F´ traD36 lacI q ∆(lacZ)M15 proA+B+/ e14– (McrA–) ∆(lac- proAB) thi gyrA96 (Nal r ) endA1 hsdR17 (r K – m K + ) relA1supE44 recA1

Restriction Enzyme Digestion > Double-stranded DNA > Two incisions, through the sugar-phosphate backbones without damaging the nitrogenous bases > Ligases; fragments from different chromosomes or genes can be spliced together  Recombinant DNA 1968; H.O. Smith, K.W. Wilcox, and T.J. Kelley isolated and characterized the first sequence specific restriction nuclease 1978 Nobel Prize – D. Nathans and H. Smith

Type I-III RE; cut outside of their recognition site Type II RE; cut inside the recognition sequence (4-8bp) – palindromic Sticky (cohesive) ends & Blunt (flush) ends EcoRI and EcoRII from Escherichia coli HindII and HindIII from Haemophilus influenzae

“four cutter” Each nucleotide has an equal chance (¼) of occurring at any particular site ¼ x ¼ x ¼ x ¼ = 1/256 A four-cutter should on average cut once every 256 base pairs.

SmaI Tango Buffer(Fermentas) 5' - CCC GGG - 3' 3' - GGG CCC - 5'

Polymerase Chain Reaction (PCR) Kary Banks Mullis 1983 / 1993 Nobel Prize in Chemistry 1986, use of DNA polymerase from thermophilic bacteria grown at a temperature of over 110°C

Thermus aquaticus / "Taq" Enzymatic halflife (at 95 o C) is 40 min Amplify a 1kb strand of DNA in roughly 60 seconds at 72°C It lacks 3'→5' proofreading exonuclease activity Error rate : 1 in 10,000 nucleotides Pwo or Pfu, from Archaea  much slower rate A (Adenine) overhangs  TA cloning

Initial denaturation o C

Template- rat retina cDNA Buffer solution - 1X Primers – µM dNTP mM Mg mM Polymerase – u/µl PCR Components DMSO (Dimethyl sulphoxide) Glycerol, BSA, PEG, Formamide

Anneal. Temp ,55056,5 Total Volume 50 µl Grp 1 (605bp) Grp 2 (635bp) Grp 3 (914bp) Grp 4 (215bp) Grp 5 (392bp) Grp 6 (1kb) Grp7 (285bp) Water31 µl Rat retina cDNA X Taq Buffer MgCl2 (25mM) Forward /Reverse primer mix(25uM) dNTP (25mM) Taq poly. (0,1u/ul)

94 o C 5 min 94 o C 30 sec Ann. T. 30 sec 72 o C 2 min 72 o C 10 min 35 cycles

* OD 260 = 2,60 * OD 280 = 1,08 a. Is your plasmid DNA pure? Why? Why not? b. Calculate the concentration (ng/ul) of your plasmid DNA and your yield if your DNA is resuspended in 50 ul water. (Your DNA was not diluted for OD measurement) c. You are going to digest 3.6 ug of your plasmid DNA by SMAI restriction enzyme. Find the missing volumes in the following chart of your digestion reaction. 10X Tango buffer 5 ul SMA I 1 ul Plasmid DNA ?? ul Dis. Water ?? ul Total 50 ul