Biol/Chem 475 Spring 2007

Transcription

1 Biol/Chem 475 Spring 2007 Goal of lab: For most of the quarter, we will be exploring a gene family that was first discovered in fruitlfies and then found to be present in humans and worms and fish and so on. The goal of this lab is to identify the widely conserved gene family that is represented by plasmid clones pct704 and pct705. These cdna clones were generated from two different genes found in the Nasonia vitripennis genome. Techniques: TNA: Visualization of total nucleic acids in an E. coli cell Preparation of plasmid DNA from E. coli strains CT704 and CT705 Dideoxysequencing of each plasmid clone (courtesy of Nevada Genomics) details of cycle sequencing will be presented in lecture Basic computational analysis of pct704&705 sequence sequence assessment, translation, alignments, BLAST, etc. Reading Assignments: Read online or in your biochem or genetics text about dideoxysequencing and about the various forms of plasmid DNA including supercoiled and relaxed circle. Schedule: Week 2 of quarter: Visualize total E. coli nucleic acids Prepare plasmid DNAs. Each pair of students will prep plasmids from both E. coli strains. Week 3 of quarter Agarose gel electrophoresis of plasmid DNA Quantitation of plasmid DNA Send to Nevada Genomics Week 4 or 5 Computational analysis of sequence DATA (details to follow..) 1

2 Week 2 of quarter: See also info in Appendix 1 E. coli strains used in this experiment CT 704 E. coli strain Top 10F transformed with pct704 CT 705 Ditto transformed with pct705 XL1Blue: contains a single copy F plasmid but not a high copy vector plasmid Genotype of TOP10 F strain of E. coli Bacterial chromosome: mcra Δ(mrr-hsdRMS-mcrBC)φ80 laczδm15* ΔlacX74 deor reca1 ara D139 Δ(ara-leu)7697 gal U gal K rpsl(str R ) enda1 nup G F factor: laci q Tn10 (tet r ) *lambdoid prophage carrying lacz mutation To do the afternoon or evening before your next scheduled lab Each pair of student should set up overnight cultures of CT704 and CT705. A colony should be inoculated into 3 ml of L broth + 50 µg/ml ampicillin (final) in a 15 ml screw-capped plastic tube. [The general rule of thumb is that the volume of broth should be 25% of the volume of the container.] Incubate at 37 o overnight with vigorous shaking. (The tube should be horizontal.) Growth for more than 16 hours is not recommended since cells begin to lyse after the culture reaches saturation. Control cultures (XL1 blues) for the TNA portion of the lab will be set up for you. 2

3 Visualize Total E. coli Nucleic Acids EAch pair of students should look at CT705, CT705 & XL1Blue cells BE careful with the phenol solution. I would wear goggles for this one. While your gel is running, start your plasmid preps 1. Place 50µl of phenol chloroform (1:1) and 10 µl of loading dye [0.25% bromophenol blue (BPB) and 40% glycerol] in a microcentrifuge tube. 2. Add 100 µl of overnight culture and vortex the mixture for 10 seconds. 3. Centrifuge for 3 min. 4. Load µl of supernatant into a gel slot that is 5 mm long X 1.5 mm wide. The gel should be about 6 mm thick and 0.8% agarose cast in 1X TBE with 0.5 µg/ml of ethidium bromide. (Note: These specifications for the gel are from the original protocol.) 5. Run gel at volts. Check gel after BPB has run about half-way down the gel bed. 6. Note: when you examine your gels, you should see more or less all categories of nucleic acids. 7. Photograph your gel and tape printout into notebook. Label carefully and determine which type of nucleic acid (RNA or DNA and general category of each) each band represents. Show analysis to CT before you leave the lab for the day. Mini-prep of plasmid DNAs. Each pair of students will prep plasmids from CT704 & CT Harvest your bacterial cells in a 1.5 ml microcentrifuge by centrifugaton at 8000 rpm (~6800g). Fill the tube to the top, centrifuge. Remove supernatant and repeat. Using a pipetman, remove all traces of the supernant from the cell pellet. 2. Follow instructions below IMPORTANT NOTES: Step1: This first step should be done in the same tube you collected the cells in. Be sure that the bacterial pellet is thoroughly resuspended before you proceed to step 2. Step 2: ABsolutely do not vortex at this step. Why not? We aren t using LyseBlue Step 5: I would pipet rather than decant Step 7: Host strain genotype on previous page Step 10: elute with water and store prep in freezer 3

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5 Qiagen Hype: Qiagen miniprep columns contain a unique silica-gel membrane that binds up to 20 µg DNA in the presence of a high concentration of chaotropic salt, and allows elution in a small volume of low-salt buffer. RNA, proteins and metabolites are not retained on the membrane. QIAprep membrane technology eliminates time-consuming phenol chloroform extraction and alcohol precipitation, as well as the problems and inconvenience associated with loose resins and slurries. High-purity plasmid DNA eluted from QIAprep modules is immediately ready to use there is no need to precipitate, concentrate, or desalt. See flow diagram of Alkaline Lysis Procedure on next page. 5

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7 Week 3 of quarter Visualization of plasmids via agarose gel electrophoresis Prepare 1% gel Prepare samples of your plasmid preps. Load various quantities including a 1/10 dilution. Quantitation of plasmid DNA by comparison to a plasmid prep of known concentration Load various quantities of a plasmid prep of known concentration probably the pgem prep you used the previous week. The concentration of the reference plasmid will determined by an A260 spec reading. Send to Nevada Genomics with instructions as to which primers to use Put the appropriate volume of your plasmids into a strip tubes or 96 well plate. We will be doing two sequencing reactions with each plasmid. The cdna insert in pct704 is 1.3 kb and the cdna insert in pct705 is ~850 bp. Figure out primers next page 7

8 What primers should we instruct Nevada Genomics to use? Each plasmid will be sequenced in both directions in other words the complementary strands will be sequenced The pct704 & 705 clones were generated using a cloning vector called pcr 2.1 Examine the map of the pcr2.1 vector that was used to generate the pct704 & 705 clones (next page). Here are the primers that Nevada Genomics Offers: Which ones should we use? 8

9 Appendix 3: Map of pcr 2.1 TOPO vector pcr 2.1 TOPO vector: sequence and restriction site information: Comments for pcr 2.1-TOPO LacZa fragment: bases M13 reverse priming site: bases Multiple cloning site: bases T7 promoter/priming site: bases M13 Forward (-20) priming site: bases f1 origin: bases Kanamycin resistance ORF: bases Ampicillin resistance ORF: bases puc origin: bases nucleotides 9

10 Appendix 1 See useful general info about growing bacterial cultures for plasmid preparation at this web site: 10