MOD2 Day 5 (Engelward)Day 5 Objectives: Extract DNA Analyze DNA by Restriction MappingStart Recombination Assay (Lipofection)Day 6 Objective: Determine the frequency of Recombination between D3 and D5 by Flow CytometryOverview for Today:1) Background and Significance: Recombination Assay 2) Review of Experimental ProceduresMiniprepRestriction AnalysisLipofection3) Experiments (Starting at 1:00)Group I: Toxic Avengers1-2 Miniprep 2-3 Restriction Digests3-4 Lipofection4-5 Load Gels Group II: Recombinators1-2 Lipofection2-3 Miniprep 3-4 Restriction Digests4-5 Load Gels Gel analysis during class is optional.Background and Significance: Recombination AssayAdapted from Sandberg and from Lodish, Molecular Cell BiologySunlightPollution &FoodOxidationNO.Cigarette SmokeBase DamageNNHNHNNH2OOHOOCH3OOOORChemical Changes“Bulky” Lesions Loss andFragmentationDNA Backbone DamageSugar DamageSingle Strand BreaksDouble Strand BreaksLoss of Heterozygosity MutantNormalRecombinational RepairTranslocationsDeletionsMutantLOHExposurePointMutationsRecombinationExposure?PROBLEM: LACK OF IN VIVO MODELSOBJECTIVE: DETECT RECOMBINATION IN MAMMALS IN VIVOII) New Tools: Recombomice!Green Fluorescence ExpressionRecombination Substrate532 bp 532 bp∆∆Pronuclear InjectionPrimers532 bp+ -Candidate FoundersGreen FL IntensityOrange FL IntensityWild-Type EarCells0% FluorescentEGFP Ear Cells99% Fluorescent 0% FluorescentMouse #8Ear CellsRecombinationFrequency:1 x 10-5Mouse #12Ear CellsFlow Cytometry()(5 x 106cells)Ear Cells from Mouse #12Recombination Frequency = ~ 3/100,000Non-Fluorescent CellsFluorescent Cells∆∆∆pCXNNX-D5∆pCXNNX-D3EGFPpCXNNX-EGFP+∆pCXNNX-EGFPParent Vector∆pCXNNX-D5What You Created!Experiment:Purify DNA from Candidates and ControlsYou will receive 4 cultures: Positive control Negative Control Two of your “PCR Hits”pCX-NNX-D5pCX-NNX??Miniprep in Short Form: Start water boiling. Spin overnight cultures for 30 seconds in an eppendorph centrifuge. Remove supernatant. Add 350 ul STET.  Poke toothpick into the lysozyme and twizzel to fullyresuspend the pellet. Boil for 40 seconds. Spin 5 minutes. Pull out yucky stuff.  Add 40 ul of 2.5 M NaAcetate (pH 5.2) and 420 ul of isopropanol. Spin 5 minutes. Pour off supernatant. Squirt 70% Ethanol into the tube to fill it. Pour off supernatant. Spin for 5 seconds in the eppendorph centrifuge. Remove the remaining small amount of ethanol. Leave horizontal on the bench for 5 minutes to let it dry. Resuspend in 40 ul of TE. Use 10 ul for each digest.What enzymes did you choose?pCXNNX-EGFPParent VectorpCXNNX-D5Mutant D5 VectorExample Digestions:Enzymes: SalI w/ and w/out EcoRVBest to use Buffer #32X Digestion Buffer:Per Reaction: For 5 Reactions:0.25 ul SalI 1.25 ul SalI2 ul Buffer #3 (stock) 10 ul Buffer #3 (10X stock)7.75 ul ddH2O 38.75 ul ddH2O------- ---------10 ul 50 ulFor 5 Reactions (one extra):SalI Alone EcoRV Alone SalI/EcoRV Neg. Control1.25 ul SalI 1.25 ul EcoRV 1.25 ul EcoRV 1.25 ul ddH2010 ul Buffer #3 (10X) 10 ul #3 (10X) 1.25 ul SalI 10 ul #3 (10X)38.75 ul ddH2O 38.75 ul ddH2O 10 ul #3 (10X) 38.75 ul ddH2O------- --------- 37.5 ul ddH2O ---------50 ul 50 ul ----------- 50 ul50 ulAdd enzymes last to these master mixes! Label tubes carefully. Then combine 10 ul of purified DNA with 10 ul of master mix.From each digest, load: 6 ul (be sure to add loading buffer!)Leave a post it note with lane labels before leaving if you don’t collect your image before you go!30 Seconds on Mammalian Cell Culture! (More in your next module…)SterilityMediaAttached vs NonadherentTrypsinizationMethods of Transfection:ChemicalPhysicalViral∆pCXNNX-D5∆pCXNNX-D3∆pCXNNX-D5∆pCXNNX-D3EGFPpCXNNX-EGFP+Invitrogen’s Lipofectamine 2000 Contents and StorageLipofectamine 2000 is supplied in liquid form at a concentration of 1 mg/ml. Store at +4°C. DO NOT FREEZE. Product is guaranteed for 6 months from the date of shipment if stored properly. Description Lipofectamine 2000 is a proprietary formulation suitable for the transfectionof nucleic acids into eukaryotic cells. Using Lipofectamine 2000 fortransfection provides the following advantages: • The highest transfection efficiency in many cell types and formats (e.g. 96-well). • DNA-Lipofectamine 2000 complexes can be added directly to cells in culture medium (in the presence or absence of serum). • It is not necessary to remove complexes or change or add mediumfollowing transfection, although complexes can be removed after 4-6 hours without loss of activity.Invitrogen’s Lipofectamine 2000 Product Qualification Lipofectamine 2000 is tested for the absence of microbial contamination using blood agar plates,Sabaraud dextrose agar plates, and fluidthioglycolate medium, and functionally bytransfection of CHO-K1 cells with a luciferasereporter-containing plasmid.Invitrogen’s Lipofectamine 2000 Important Guidelines Follow these guidelines when performing transfections: 1. The ratio of DNA (in µg):Lipofectamine. 2000 (in µl) to use when preparing complexes should be 1:2 to 1:3 for most cell lines. 2. Transfect cells at high cell density. 90-95% confluence at the time oftransfection is recommended to obtain high efficiency and expression levels,and to minimize decreased cell growth associated with high transfectionactivity. Lower cell densities are suitable with optimization of conditions. Take care to maintain a standard seeding protocol between experiments because transfection efficiency is sensitive to culture confluence. 3. Do not add antibiotics to media during transfection as this will cause cell death.Invitrogen’s Lipofectamine 2000 For optimal results, we also recommend the following: 1. Use Opti-MEM® I Reduced Serum Medium (Catalog no. 31985-062) to dilute Lipofectamine. 2000 prior to complexingwith DNA.Invitrogen’s Lipofectamine 2000 Transfection Procedure Use the following procedure to transfect mammalian cells in a 24-well format. Up or Down Transfections. 1. Adherent cells: One day before transfection, plate 0.5-2 x 10^5 cells in 500 µl of growth medium without antibiotics per well so that they will be 90-95% confluent at the time oftransfection.Invitrogen’s Lipofectamine 2000 2. For each transfection sample, prepare DNA-Lipofectamine 2000 complexes as follows: a. Dilute DNA in 50 µl of Opti-MEM® I Reduced Serum Medium without serum (or other medium without serum). Mix gently. (BE109: You will be given DNA