Procedure: GFP cloning project Day 1. Bioinformatics and cloning workshop. Agar plate prep. 1. Prepare nutrient agar (required in next few labs for bacterial work). a. To prepare the agar, weigh 3.85g LB-agar and pour into a 250ml Erlenmeyer flask. Luria Broth (LB) is a nutritionally rich medium suitable for bacterial growth; it contains tryptone, yeast and NaCL. Agar is a gelling agent that solidifies the mixture at room temperature. b. Add 110 ml of distilled water to a 250 ml Erlenmeyer flask. Cover the top with foil and label with your name and section using a small piece of tape on top of the foil. Use a permanent marker. c. Leave on center bench for the technician to autoclave (sterilize). Your LB-agar will sterilize at 121oC for 20 minutes. It will be ready to pour later during lab. Bioinformatics This portion of the lab will be performed in the computer lab. Bioinformatics is the application of computer technology to the storage and use of biological data, including protein and nucleic acid sequence and structural information. There are a plethora of websites containing databases, directories, catalogs and tools of interest to biochemists and molecular biologists. Below are listed but a few: Pedro s Biomolecular Research Tools (A Collection of WWW Links to Information and Services Useful to Molecular Biologists) http://www.biophys.uniduesseldorf.de/bionet/pedro/research_tools.html CMS Molecular Biology Resources (A compendium of electronic and internet accessible tools and resources for Molecular Biology and Biochemistry) http://mbcf.dfci.harvard.edu/cmsmbr/ Protocol Online (Lab reference book) http://www.protocol-online.org/ GenBank (The NIH genetic sequence database, an annotated collection of all publicly available DNA sequences), http://www.ncbi.nlm.nih.gov/genbank/ EMBL-EBI (databases of biological data including nucleic acid, protein sequences and macromolecular structures.) http://www.ebi.ac.uk/ Protein Data Bank, PDB (Protein structures determined by X-ray and NMR) http://www.rcsb.org/pdb/home/home.do Swiss-protein (Protein sequences and analysis) http://ca.expasy.org/tools/ AAindex (Properties of amino acids) http://www.genome.jp/aaindex/ BLAST (The Basic Local Alignment Search Tool finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families.) http://blast.ncbi.nlm.nih.gov/blast.cgi Chime (Chime is a browser plug-in that renders 2D and 3D molecules directly within a Web page. The molecules are "live", meaning they are not just static pictures, but chemical structures that scientists can rotate, reformat, and save in various file formats for use in modeling or database applications.) http://www.mdl.com/products/framework/chime/
For a more comprehensive list of websites please refer to: Biochemistry Laboratory, modern theory and techniques. Rodney Boyer. ISBN 0-8053-4613-9 Benjamin Cummings press. Computational exercise (Cloning workshop): determining GFP gene sequence and PCR primer design. 1. Using the website EMBL-EBI, download and print the nucleotide sequence for Green florescent protein. Website: http:/www.edi.ac.uk/databases/ You will be searching for GFP, then click on Nucleotide sequences. Scroll down to results found in EMBL-Bank (Coding Sequence). 2. Determine what restriction enzyme sites are present within the GFP gene. Use the following website: http://www.bioinformatics.org/sms2/rest_map.html. Print your results. 3. Observe and print the X-ray crystallography structure of GFP using the Protein data bank. Use the following website and search for GFP: http://www.rcsb.org/pdb/home/home.do View the most recent citation. Click on view in Jmol, hold the left mouse key down and drag to rotate the molecule and view in different orientation. 4. Design primers for amplification of the GFP gene by PCR. Use http://www.bioinformatics.org/sms/rev_comp.html to determine the reverse complement for the 3 primer. Use http://www.sigmaaldrich.com/configurator/servlet/designtool?prod_type=standard to determine the technical data on the primers you have designed and determine if they are appropriate for PCR.
Discussion: Generation of a GFP expressing plasmid (pglo) using the GFP PCR fragment and cloning it into an expression vector. pglo, 5.37Kb Return to the lab. Prepare the agar plates (you will use them for the transformation step in the next lab period): 1) The agar you prepared last lab period has been autoclaved (sterilized at 121oC for 20 minutes and pressure (~15psi)). While you are waiting for the agar to cool, label the plates. The agar plates should be marked with a permanent marker on the bottom close to the edge. Label each plate with your name and date. Label 1 plate LB, 1 plate LB/amp and 2 plate2 LB/amp/ara. 2) Sterile solutions of Arabinose (200mg/ml) and Ampicillim (10mg/ml) are provided. When adding these reagents to your LB-agar use sterile technique (your TA will demonstrate how). Sterile tips must be used, be careful not to contaminate the stocks or your LB-agar. Note: Excessive heat (>50 C) will destroy the ampicillin and the arabinose, but the nutrient agar solidifies at 27 C so one must carefully monitor the cooling of the agar and then pour the plates from start to finish without interruption. Excess bubbles can be removed after all the plates are poured by briefly flaming the surface of each plate with the flame of a Bunsen burner. After the plates are poured do not disturb them until the agar has solidified. Pour excess agar in the garbage, not the sink. Wipe any agar drips off of the sides of the plates.
Pour LB nutrient agar plates (LB, LB/amp, LB/amp/ara) YOUR TA WILL SHOW YOU HOW. 3) First, pour LB nutrient agar into the plate labeled LB. 4) Add 850ul of the ampicillin (10mg/mL) to the remaining LB nutrient agar. 5) Swirl briefly to mix. 6) Pour 25mL to the plate labeled as LB/amp using the technique utilized above. 7) Add 2mL of the arabinose to the remaining LB nutrient agar containing ampicillin. 8) Swirl briefly to mix and pour the plates labeled as LB/amp/ara using the technique utilized above. 9) LB broth. Place 2g LB broth into a 250ml Erlenmeyer flask, add 100mls ddh2o. Cover top with foil and label with your name. Leave on community bench for the technician to autoclave for use later on. 10) Place 4-5 eppendorf tubes in a 10mL beaker, cover with foil. Label and place on community bench. Interim Day: Place agar plates in a labeled plastic bag and store upside down in the cold room. They are stored upside down to prevent condensation dripping onto the agar. Day 2.Transformation. Collect your sterilized LB broth. We will provide the GFP plasmid for transformation. Each student should: 1. Label one closed sterile eppendorf tube +pgfp and another -pgfp. Label both tubes with your name. 2. Open the tubes and, using a sterile pipette, transfer 250 μl of transformation solution (50mM CaCl2) into each tube. 3. Place the tubes on ice. 3. Use a sterile loop to pick up a single colony of bacteria from your starter plate. Pick up the +pgfp tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tube back on ice. Using a new sterile loop, repeat for the -pgfp tube. 4. Pipette 1uL of the GFP plasmid to the +GFP tube, mix gently with the pipette tip. Close the tube and return it to the ice. Also close the -pgfp tube. Do not add plasmid DNA to the - pgfp tube. 5. Incubate the tubes on ice for 10 minutes. 6. While the tubes are sitting on ice, label your four LB nutrient agar plates on the bottom (not the lid) as follows: Label one LB/amp plate: + pgfp Label the LB/amp/ara plate: + pgfp Label the other LB/amp/ara plate: - pgfp Label the LB plate: + pgfp 7. Heat shock. Using a foam rack as a holder, transfer both the (+) pgfp and (-) pgfp tubes into the water bath, set at 42 C, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water. When the 50 seconds are done, place both tubes back on ice. For the best transformation results, the transfer from the ice (0 C) to 42 C and then back to the ice must be rapid. Incubate tubes on ice for 2 minutes. 9. Remove the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipette, add 250 μl of LB nutrient broth to the tube and re-close it. Sterile technique requires that you pass the top of the LB broth
through a flame prior to pipetting. Repeat with a new sterile pipette for the other tubes. Incubate the tubes for 10 minutes at room temperature. 8. Tap the closed tubes with your finger to mix. Using a new sterile pipette for each tube, pipette 100 μl of the transformation and 100 μl control suspensions onto the appropriate nutrient agar plates. 11. Use a flamed sterilized loop for each plate. Spread the suspensions evenly around the surface of the LB nutrient agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface (your TA will demonstrate how). DO NOT PRESS TOO DEEP INTO THE AGAR. 9. Let solution absorb onto plates for ~15 minutes. 10. Stack up your plates and tape them together. Put your name and class period on the bottom of the stack and place the stack of plates upside down in the 37 C incubator until the next day. 11. Prepare 200mls of TE buffer. 12. Prepare 10mls each of column equilibration buffer and column wash buffer using the calculations from your pre-lab. Store in cold room for later use. Prep your culture tubes for interim day: 13. Obtain two 15ml sterile culture tubes. Mark with your name. 14. Using sterile technique, pipette 3mL LB broth into each tube. 15. Pipette the amount of ampicillin you calculated in your pre-lab into each tube. 16. Pipette the amount of L-arabinose you calculated in your pre-lab into each tube. 17. Store in cold room for use in the interim day. Interim day 1. Examine your LB/amp and LB/amp/ara plates from the transformation lab. First use normal room lighting, and then use an ultraviolet light in a darkened area of your laboratory. Note your observations. To prevent damage to your skin or eyes, avoid exposure to the UV light. Never look directly into the UV lamp. Wear safety glasses whenever possible. 2. Identify several green colonies that are not touching other colonies on the LB/amp/ara plate. Turn the plate over and circle several of these green colonies. On the other LB/amp plate identify and circle several white colonies that are also well isolated from other colonies on the plate. 3. Obtain the two 15 ml culture tubes that you prepared in the previous lab. Using a sterile inoculation loop, lightly touch the "loop" end to a circled single green colony and scoop up the cells without grabbing big chunks of agar. Immerse the loop in one culture tube. Spin the loop between your index finger and thumb to disperse the entire colony. Repeat for the other culture tube. Streak an Amp/Ara plate with the loop and place in incubator. 4. Cap your tubes and place them in the shaking water bath. Let the tubes incubate for 24 hours at 32 C. Day 3. Plasmid Preparation and analysis. 1. Retrieve culture tubes from the shaking water bath. 2. Observe florescence with UV lamp. 3. Place tubes in the clinical centrifuge (be sure to balance the centrifuge by placing the two tubes in opposite positions) and spin at top speed for 15 minutes. 4. Carefully pour off liquid.
5. Resuspend each pellet in 1 ml ddh2o and transfer to two 1.5ml eppendorf tubes. Spin for 2 minutes at 10,000xg to pellet cells. 6. Pipette off supernatant and discard. 7. Resuspend each pellet in 200ul resuspension solution. 8. Add 200ul cell lysis solution, mix well. 9. Add 200ul neutralization solution, mix well. 10. Centrifuge at 10,000xg for 5 minutes. 11. Assemble two minicolumns each with a 3ml syringe barrel. Shake resin well and add 1ml resin. 12. Transfer the supernatant from step 12. to the two columns. 13. Insert syringe plunger and press to eject liquid (do not save). 14. Remove column from syringe. 15. Remove plunger. 16. Re-attach column to syringe. 17. Add 2mls column wash solution to each syringe. 18. Insert syringe plunger and press to eject liquid (do not save). 19. Remove syringe barrel and place each column in a 1.5ml eppendorf tube. 20. Centrifuge at 10,000xg for 2 minutes (be sure to balance the centrifuge with another students sample). 21. Transfer each minicolumn to a new eppendorf tube. 22. Add 30ul sterile water to each and wait 1 minute. 23. Centrifuge at 10,000xg for 20 seconds. Restriction enzyme analysis. 1. Using the pglo plasmid map notes from your pre-lab set up a suitable diagnostic enzyme digest for your plasmid prep. Incubate at 37oC for 1 hour and then at 65oC for 15 minutes. a. 15uL plasmid b. 2uL buffer (see chart for appropriate buffer) c. 1uL BSA (from diluted stock) d. 1uL sterile water e. 1uL restriction enzyme 2. Pour a 1% DNA agarose gel. 3. Load gel with digest samples and ladder. Run at 100V for 40 minutes. 4. Pour 50mL 1x Sybr gold solution into staining tray. Add gel. 5. Cover with foil and agitate for 10 minutes. 6. View and photograph gel. Prepare culture tubes and set up cultures for protein purification. 1. Obtain two 15ml sterile culture tubes. Mark with your name. 2. Using sterile technique, pipette 3mls LB broth into each tube. 3. Pipette the amount of ampicillin you calculated in your pre-lab into each tube. 4. Pipette the amount of L-arabinose you calculated in your pre-lab into each tube. 5. Inoculate each tube with a GFP expressing colony.