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UW Igem Lab Training Exercise iGEM Training UW iGEM Lab Training Exercise The overall goal of the lab exercise is to transform E. coli cells previously unable to express GFP (green fluorescent protein), to now express GFP. GFP expression is visible when the cells are viewed under UV light. This is done by turning on a gene, which is achieved by positioning a promoter in front of the sequence and assembling it into another vector. This is done all in one step. (Drawn on board) To separate out the plasmid DNA (which acts as the vector to contain our gene of interest) we use the cells inherent chemical nature and manipulate it to differentiate the cell components. Centrifuging is a big component of the separation of cell components by spinning at high speeds. It separates components of different sized in a mixture and is therefore used to separate out different cell components. Important Notes on Centrifugation… (When centrifuging make sure that tubes are placed across each other so that the centrifuge is balanced. If you have an odd number of tubes, a microfuge tube filled with the equivalent amount of water can be used as a “dummy tube”. Orient the tube with the hinge facing up so you can anticipate where the pellet is going to form, as it may be difficult to see a pellet in some miniprep procedures) MOST IMPORTANTLY!: This is a sequential process and the samples that you are working with will be carried on to the next day. If at any point you are confused/unsure ASK! CONTENTS DAY 1: Pipette Basics, Plasmid Miniprep and Restriction Digest .............................................................................2 Use of Micropipettes ........................................................................................................................................2 Supplementary Exercise: Standard Curve of Fast Green ..................................................................................2 Plasmid Miniprep ..............................................................................................................................................4 Measuring the Concentration and Purity of DNA.............................................................................................5 Restriction enzyme digestion ...........................................................................................................................6 DAY 2: Agarose Gel Electrophoresis and Ligation ....................................................................................................8 Making a Gel .....................................................................................................................................................8 Setting up and Loading the Gel ........................................................................................................................9 Purification protocol (to separate DNA from Restriction Enzymes)(BioBasic): ............................................. 10 Ligation Reaction ........................................................................................................................................... 11 1 iGEM Training DAY 3: Transformation .......................................................................................................................................... 12 Cell Transformation using Heat Shock ........................................................................................................... 13 Plating ............................................................................................................................................................ 13 Materials ........................................................................................................................................................ 13 DAY 4: Results ........................................................................................................................................................ 15 Nanodrop ............................................................................................................................................................... 15 DAY 1: PIPETTE BASICS, PLASMID MINIPREP AND RESTRICTION DIGEST Use of Micropipettes This is a short exercise to familiarize you with what µL volumes look like in microfuge tubes. It is a good habit to anticipate what you expect in terms of volume so that you know if all the components of your reaction have been added (and to ensure that your pipette is properly calibrated). Materials - P10 Micropipette - Micropipette tips - 5 microfuge tubes Into separate microfuge tubes, pipette 10.0, 5.0, 2.0, 1.0 and 0.5µL of water using a P10 micropipette. (Pay special attention to how volumes look in pipette. Note that the pipette is conical, so lower amounts don't look proportionally as small in the tip as you'd expect.) Note that it is common to indicate a decimal place when calibrating a pipette by a line or a change in font color. Always look at the top of the pipette to see the volume range it can accommodate. Supplementary Exercise: Standard Curve of Fast Green One of the goals of this additional exercise is to test the precision and reproducibility of your micropipetting skills by using the Genesys spectrophotometer to measure absorbance of a fast green dye to produce a standard curve. 2 iGEM Training If the absorbance values of the two different samples of the same concentration are the same (or very close) then you know that your pipetting is precise. Materials - Fast green solution - dH2O - 18 microfuge tubes - 3 cuvettes Protocol: 1. Before you begin, turn on the Genesys spectrophotometer and set the wavelength to 635 nm. There will be an instruction sheet on how to use the spectrophotometer next to each unit. Final concentration Volume of Fast Volume of dH20 OD 635 OD 635 of Fast Green Green diluent Sample 1 Sample 2 @ 10 µg/ml Blank 0.1µg/ml 0.25 µg/ml 0.5 µg/ml 1.0 µg/ml 100 µl 900 µl 2.5 µg/ml 5.0 µg/ml 7.5 µg/ml 10.0 µg/ml 2. Using the stock solution of Fast Green provided (10 g/ml) and dH2O as your diluent, prepare samples for a standard curve in duplicate (You will need two samples for each concentration). This will be done by diluting the stock to the following concentrations; 0.1 g/ml 0.25 g/ml, 0.5 g/ml, 1.0g/ml, 2.5 g/ml, 5.0 g/ml, 7.5 g/ml and 10 g/ml in 1.5 ml microfuge tubes. The final volume of each dilution should be 1.0 ml 3. Vortex all tubes to mix thoroughly. 4. Use 3 disposable cuvettes in total to take your readings. One will be for your dH2O blank, the other two for each of your duplicate samples. 5. Begin by zeroing the Genesys spec with the dH2O blank. 6. Starting with the most dilute Fast Green samples, transfer the tube contents to the two cuvettes and read the absorbance at OD635. Record the values in the table provided above. 3 iGEM Training 7. Return the contents of the cuvette to the appropriate microfuge tube and rinse the cuvette thoroughly with dH2O and transfer the contents of the next dilution series to the cuvettes to read. 8. Continue with the increasing concentrations of Fast Green until all samples have been read and recorded in the table. Plasmid Miniprep A plasmid miniprep is a procedure used to isolate plasmids from the cells that we are interested in. Materials - 3 cultures - 6 microfuge tubes - 3 spin columns 1. Acquire tubes with cultures, 1 of each: The resuspension DH5α-BBa_J61002-J23101 (iGEM strain #72), solution is just buffer in DH5α-pSB1A2-E0240 (iGEM strain #110), which your cells now DH5α-pSB1C3-J04450 (iGEM Strain #111). float. Also it contains RNase, which will chop 2. Acquire 3 microfuge tubes. Label to differentiate the three cultures up unnecessary RNA (strain number is probably the best since it is the smallest identifier) and when cells are broken your initials. up. 3. Pipette 1.5 mL of each culture into a different microfuge tube and centrifuge the three tubes at 12,000rpm for one minute. Discard the Performing steps 4 and supernatant (liquid). 5 allows you to collect 4. Into the same microfuge tubes with the pellet, pipette 1.5ml of the more cells and give you a larger amount of DNA cultures and centrifuge the three tubes at 12,000 rpm for one minute. Discard the supernatant *To aspirate means to 5. Repeat step 4. take up the volume of 6. Add 100µL of resuspension solution with a P200 pipette, and gently the solution and pellet aspirate* to mix. Wait about a minute. with the tip and then 7. Add 200µL of lysis buffer with P200. Invert the tube gently 5 times. expel it. Repeat this Incubate tube on ice for 1-3 minutes until the pellet is dissolved) 8. Add 350µL with P1000 of neutralization solution. Allow to incubate at room temperature for 1 minute. The lysis solution is 9. Spin at 12,000 rpm for 5 min. alkaline and it causes At this step if your pellet has not formed neatly (aka it is loose and cloudy breaking up the cells. along the side) the tube should be spun at 12,000rpm for another 2 The contents of the cell minutes. It sometimes helps to rearrange the tubes in the centrifuge if lose their original state 4 iGEM Training some of the pellets are properly formed but others are not. and precipitate out of 10. Obtain and label 3 spin columns. Use P200 micropipette and pipette out solution 200 µL of the supernatant at a time into the spincolumn. (Take special care to avoid area of pellet) The volume (and pipette tip) can be adjusted for optimal extraction. The addition of neutralization solution causes the DNA to renature. When the genomic DNA renatures it does not do so neatly and the larger gel like formation that
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