Kariuki practical report

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2. A. DNA TECHNIQUES1.0 CLONING NANOBODY GENE1.1 INTRODUCTIONThe gene is the corner stone of most molecular biology techniques. It is possible today toamplify a gene through insertion in another DNA molecule that serves as a vehicle or vector thatcan effectively divide inside living cells (Watson, 2007). The most widely used vectors includebacterial plasmids, Cosmids and phages. This is a recombinant DNA molecule that is theninserted inside a prokaryotic or eukaryotic cell as host. As the host cell replicates, the vectortogether with the inserted foreign DNA also replicates. Through this the foreign DNA becomesamplified in number and further analysis can be performed.Insights into recombinant DNA technology came from among others the observation of theability of the linear genome of lambda phage DNA to circularize when it enters the host bacteriacell by Allan Campbell in 1962. Further analysis revealed that lamda phage had short regions ofsingle stranded DNA whose base sequences were complementary to each other at the end of itslinear genome referred to as cos sites (cohesive sites). Further insights came fromcharacterization of bacterial restriction/ modification systems by Salvador Luria and other phageworkers which became apparent bioengineering tools for creating cohesive ends throughrestriction endonucleases.Cloning vectors are the carrier of the DNA molecule and all vectors must have some importantfeatures which include capability to independently replicate themselves and the foreign DNAthey carry, secondly, they must contain a number of restriction endonuclease cleavage siteswhich are present only once in the vector. Thirdly they must carry a selectable marker usuallyinform of an antibiotic resistance to distinguish host cells that carry the vector from host cellsthat do not carry the vector. Finally, they must be relatively easy to recover from the host cell.Transformation is the process of introducing the ligation mixture of recombinant and non-recombinant DNA into the host bacterial cell. A mutant bioengineered strain of E.coli bacteriadeficient of restriction modification system is used. The traditional method of transformationinvolve incubating the bacteria in a concentrated calcium salt overnight to make their membraneleaky but a more efficient method involve heat shock treating or electroporation.Amplification is done by Polymerase Chain Reaction, PCR developed by Kary Mullis in 1985.With PCR, a single segment of DNA can be amplified a billion times in several hours. Theprocedure is carried our entirely in vitro through three important processes. Since it is a DNApolymerase reaction it requires a DNA template and a 3 OH provided by the DNA sample to beamplified and the site-specific oligonucleotide primers respectively. The three steps of thereaction are denaturation, annealing of the primers and extension of the primers. Denaturation isthe first step done by heating the double stranded DNA to make single stranded DNA template.Annealing is done by cooling to allow the primers to bind to the appropriate complementary 3. strands. Primer extension is the last step and happens in the presence of magnesium ions andDNA polymerase which extends the primers on both strands from 5 3 direction. Currently themost popular DNA polymerase is Taq polymerase from thermophilic bacteria Thermusaquaticus. Temperature variations is done using an instrument called a thermo cycler with thecapability of rapidly switching between the different temperatures that are required for PCRreactionVisualization of PCR products is done on a gel stained with a nucleic acid-specific fluorescentcompounds such as ethidium bromide or SYBR green.2.0 MATERIALS AND METHODS2.1 PLASMID ISOLATION2.1.1 MATERIALS Sterilized 2ml eppendorf tubes Sterilized micropipette tips Sterilized bacterial cultures Ice in box Centrifuge Luria broth (LB) media (liquid) Cell re-suspension solution (P1) Cell lysis solution (P2) Neutralization solution (P3) Isopropanol (kept at -20oC) 70% (v/v) ethanol, (kept at -20oC) Sterilized water Waste beaker/ dissinfectants E.coli call WK 1168 containing PHEN6c2.1.2 PROCEDUREAn E. coli suspension incubated the previous night in 5ml LB containing ampicillin in a 50mltube at 370C was harvested and 1.5ml pelleted via centrifugation at 12,000xg for I minute. Thesupernatant was discarded. The bacteria pellet was then re-suspended with 200l of the re-suspension solution (P1). Vortexing was done to thoroughly re-suspend the cells untilhomogeneous. The re-suspended cells were lysed by adding 200l of the lysis solution (p2). The contents wereimmediately mixed by gentle inversion 7 times until the mixture became clear and viscous. Thelysis reaction was not allowed to exceed 5 minutes. The cell debris was then precipitated byadding 350l of neutralizing solution (P3). The tube was gently inverted 6 times. The cell debris 4. was pelleted by centrifuging at 12,000xg for 10 minutes for cell debris, proteins, lipids, SDS andchromosomal DNA to fall out of solution as cloudy, viscous precipitate.The column was prepared by inserting the GenElute Miniprep Binding column into a providedmicro-centrifuge tube. 500l of the column preparation solution was added to each miniprepcolumn and centrifuged at 12,000xg for 1 minute. The flow-through liquid was discarded. Thecolumn preparation solution was meant to maximize the binding of DNA to the membraneresulting in more consistent yields.The cleared lysate from neutralization reaction above was transferred to the prepared column andcentrifuged at 12,000xg for one minute and the flow-through liquid discarded. 500 of optionalwash solution was added to the column and centrifuged at 12,000xg for one minute and the flow-through liquid discarded. 750l of the diluted ethanol wash solution was added to the columnand centrifuged at 12,000xg for 1 minute. This column was step removes residual salt and othercontaminants introduced during the column load. The flow-through liquid is discarded andcentrifuged again at maximum speed for 2 minutes without additional wash to remove excessethanol.To elute DNA, the column was transferred to a fresh column and 100l of elution solution addedto the column followed centrifuging at 12,000xg for 1 minute.The purified plasmid DNA was present in the elute and its concentration was determined usingthe NanodropTM. The elute was stored at -200C.2.2 RESTRICTION ENDONUCLEASE DIGESTION OF NANOBODY GENE ANDPHEN6c2.2.1 Materials PCR fragment (Nanobody gene) PHEN6c plasmid 10x O-buffer (fermentas) PCR clean up kit-GenElute Spectrophotometer/ NanodropTM Water bath (370C) Eco 911 (fermentas, 10 units/l) Pstl (fermentas, 10 units/l) Ethanol Micro centrifuge Micro centrifuge tubes Molecular Biology water 5. 2.2.2 Procedure2.2.2.1 Digestion of vector for ligation and checking size.Digestion was done by taking 10q plasmid + 5l O-buffer (fermentas) + 1l PstI (fermentas, 10units/l) + 1l Eco911 (fermentas, 10 units/l) and topped up to 50l. into another tube digestfor checking vector size was digested with only Pstl restriction enzyme and incubating for twohours at 370C was performed. Checking digestion and vector size was done the following dayusing 0.8% agarose. Digests for ligation were purified as follows.The GenElute plasmid mini spin column is inserted into a provided collection tube. 0.5ml of thecolumn preparation solution was added to each mini spin column and centrifuged at 12,000xg for1 minute. The elute was discarded. The column preparation solution maximizes binding of DNAto the membrane resulting in more consistent yields. 250l of the binding solution was added tothe 50l of the plasmid DNA. The solution was transferred to the binding column andcentrifuged at 12,000xg for one minute. The elute was discarded but the collection tube wasretained.The binding column was replaced into the collection tube.0.5ml of the diluted wash solution isapplied to the column and centrifuged at a maximum speed for one minute. The elute isdiscarded but the collection tube retained. The column is replaced in the collection tube andcentrifuged at maximum speed for 2 minutes without any additional wash solution to removeexcess ethanol. The residual elute and as well as the collection tube were discarded. The columnwas then transferred to a fresh 2ml collection tube. 50l of elution solution was applied to thecentre of the column and incubated at room temperature for 1 minute. The column was thencentrifuged at maximum speed for 1 minute. The Plasmid DNA was available in the elute and itspurity concentration was determined by NanodropTM.The same procedure was followed by the other group to purify the PCR fragment (nanobodygene).2.3 LIGATION2.3.1 Materials Eppendorfs Digested vector and PCR fragment Water bath dH2O T4DNA ligase (5units/l) 10 ligation buffer 6. 2.3.2 ProcedureLigation was done by mixing 50ng vector + 50ng PCR fragment (1:1) + 2l 10x ligation buffer +1l T4 DNA ligase (5units/l) and filled up with dH2O until the end volume was 20l. All thethree tubes were incubated for 1 hour at room temperature.2.4 HEAT SHOCK TRANSFORMATION2.4.1 Generation of CaCl2 competent E. coli cells2.4.1.1 MaterialsReagents LB medium Sterile ice cold 0.1M MgCl2 Sterile ice cold 0.1 100% glycerol Fresh E.coli WK6 403 strainEquipment 50ml blue caps Sterile 1.5ml eppendorfs Shaking flask with baffles Cooled centrifuge for 50ml tubes Laminar air flow Spectrophotometer and cuvettes2.4.1.2 ProceduresFive milliliters of LB (without antibiotics) is prepared in one sterile 50ml tube. The tube is theninoculated with a single colony of E.coli WK 403 from a fresh plate. It was the tube wasincubated at 370C. The culture was harvested the following