APh 162 – Molecular BiologyDay 2Restriction DigestsThe LacZ gene on our plasmid is flanked by restriction sites for the enzyme `KpnI’ on one side and `HindIII’ on the other, allowing us to cut the gene out and leave precisely controlled ‘sticky-ends’ on the plasmid DNA, onto which we will ligate our new reporter gene (or `insert’), called -Venus(YFP). The enzyme KpnI cuts at any site with the sequence ...5-GGTAC’C-3... leaving the sticky-end GTAC, while the enzyme HindIII cuts at any site with the sequence ...5-A’AGCTT-3... leaving the sticky-end AGCT. The fact that these sticky ends are distinct means we can discourage the plasmid from ligating on itself, and ensure that we insert the new reporter gene in the correct orientation. A myriad of available restriction enzymes ensures that we can control each piece of the plasmid independently. In today’s lab session, we will digest pZE21-lacZ and the YFP (Venus) PCR amplicon from day 1.Restriction enzyme concentrations are measured in the units of `activity’, where 1 unit of enzyme can digest 1 g of DNA in 1 hour at 37C. To ensure that all of the DNA is cut by the enzyme, it is generally recommended to add enzyme in excess of the required amount.Follow the protocol below, and mix the following reagents to begin this enzymatic reaction. Once the reaction is finished, we will examine the products on an agarose gel to determine their size and differences in topology. To properly gauge the size of DNA segments that appear on the agarose gel, we need to add a so-called `DNA Ladder’ that contains segments of known length, against which we will compare our plasmid digest. We will use two commercially available ladders, and we will create our own `DNA Ladder’ using the distinct enzyme EcoRI to digest –phage (viral) genomic λDNA that has been pre-digested with HindIII. Today you will perform the following restriction digests:1. KpnI/HindIII double digest of the purified, PCR-generated YFP (Venus) insert from Day 1.2. KpnI single digest of vector pZE21-LacZ3. HindIII single digest of vector pZE21-LacZ4. KpnI/HindIII double digest of vector pZE21-lacZ5. EcoRI single digest of pre-HindIII digested lambda phage DNADouble Digest of YFP (Venus) insert Reagent: Amount:YFP DNA (purified, from Day 1 PCR) 200 ngNEB Buffer 2 (10X)(NEB-B7002S) 1XBSA (10X) 1XKpnI (10 units/l) 2 lHindIII (10 units/l) 2 lDDH2O Calc.Total Volume 50 lDouble Digest of pZE21-lacZ vector - Reagent: Double Digest:Kpn I Single DigestHind III Single DigestNo Digest ControlPlasmid (pZE21_LacZ ) DNA* 300 ng 300 ng 300 ng 300 ngNEB Buffer 2 (10X) 1X 1X 1X 1XBSA (10X) 1X 1X 1X 1XKpnI (10 units/ul) 1 l 1 l 0 l 0 lHindIII (10 units/ul) 1 l 0 l 1 l 0 lDDH2O Calc. Calc. Calc. Calc.Total Volume 30l 30l 30l 30l* Your TA will provide the concentration. – phage Digest Reagent: Amount:Digested DNA 0.5 ug/λ l 1.5 gNEB EcoRI Buffer (10X) 1XBSA (10X) 1XEcoRI (10 units/l) 2 lDDH2O Calc.Total Volume 30lNotes: NEB Buffer 2 is one of many potential buffers, chosen to optimize the performance of both KpnI and HindIII. See “Enzyme Properties” at: http://www.neb.com/nebecomm/products/productR0104.asphttp://www.neb.com/nebecomm/products/productR0142.aspProcedure:1. Verify all calculated reagent volumes with your TA.2. For the given concentration of input DNA, calculate the corresponding volume.3. Mix this volume of DNA along with the specified amounts (see tables above) of buffer, restriction enzymes and water to bring the total volume to 25 l, in a small tube. When finished, ask your TAs for the appropriate amounts of restriction enzyme 4. Mix thoroughly and spin in the micro-centrifuge for a few seconds (<6 sec) to sediment all the contents.5. In addition to using commercial ladders, we will create our own ladder using lambda phage DNA.a. The -phage genome is 48,502bp, if the sequence was totally random, how λmany HindIII (5A’AGCTT3) and EcoRI (5G’AATTC3) sites would you expect? How many are there in reality? i. Given the actual restriction sites for EcoRI and HindIII, calculate the expected segment lengths of the DNA ladder.ii. See the attached restriction site map for the -phage genome. λ6. Place all six of your tubes in a 37C incubator for 1 hour. During this incubation, we will prepare the agarose gel.DNA Agarose Gel ElectrophoresisThe above enzymatic reactions and accompanying controls have produced a range of DNA lengths and the no-enzyme (uncut DNA) control even has a distinct polymer topology. It is immensely useful to be able to measure the lengths produced during DNA manipulations. The primary method employed throughout molecular biology is to subject DNA in an ultra-viscous agarose gel to an electric field, wherein we utilize the ~-5.88e/nm charge on DNA to ‘pull’ it through the gel. In a given period of time the DNA migrates through the gel roughly proportional to the inverse of its mass. 1X TAE Buffer ~500 ml1X TAE- 1% agarose gel ~ 40 ml (40 mL TAE + 0.4g agarose)100bp DNA Ladder (NEB-N0467S) 2 l ladder + 3 l H2O + 1 l 6x loading dye1000bp DNA Ladder (NEB-N3232) 2 l ladder + 3 l H2O + 1 l 6x loading dyeFor plasmid digest, 3 controls,  DNA 25 l of digest + 5 l 6x loading dyeNote: TAE (Tris-acetate EDTA) bufferMaterials:gel box gel power supplygel tray gel combYou will run the following samples on the gel:1. 100 bp DNA ladder2. Non-purified, non-digested PCR product from day 13. Lambda phage double digest4. pZE21-LacZ – KpnI single digest5. pZE21-LacZ – KpnI single digest6. pZE21-LacZ – KpnI/HindIII double digest7. pZE21-LacZ – no enzyme control digest8. 1 kb DNA ladderProcedure:b)a)DNA Laddersa) Example of 1 kb DNA ladder on a 0.8% agarose gel, stained with EtBr.b) Example of the 100bp DNA ladder on a 1.3% agarose gel, stained with EtBr.1. Using DI water, clean the gel box, gel comb and gel tray.2. In short bursts of ~10s, heat the 1X TAE- 1% agarose gel in the microwave until it is completely liquefied.3. Secure the gel comb in the gel tray and orient so that the red rubber seals form a liquid-tightdish. Pour ~35ml of the liquid agarose into the gel tray. Be careful not to introduce any bubbles. 4. Allow the gel to solidify over the next 30 minutes, without disturbing it.5. Once fully solidified, carefully remove the gel comb by pulling it straight up, taking care not to damage the