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Team:UEA-JIC Norwich/Methods
From 2011.igem.org
HIGH EFFICIENCY PROTOCOL
1. Thaw a tube of NEB 5-alpha Competent E.coli cells on ice for 10 minutes
2. Mark the location of the Biobrick well (letters from top to bottom, numbers from left to right)
3. Resuspend specific biobrick part (1 μl) with distilled water (20 μl). Aspirate up and down a few times
4. Inoculate with DNA ( 1 μl) to the competent cells
5. Place the mixture on ice for 30 minutes. Do not mix
6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix
7. Place on ice for 5 minutes. Do not mix
8. Pipette 950 μl of room temperature SOC into the mixture
9. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate
10. Warm selection plates to 37°C
11. Do serial dilutions (2-3) at 105. Mix cells, flick/invert gently
12. Spread (100 μl) on agar plates of each dilution
13. Incubate overnight at 37°C
CULTURE PREPARATION PROTOCOL
1. Select a colony by sampling it with a wooden pick
2. Place into the appropriate antibiotic LB broth
3. Incubate at 37°C overnight in an Incubator Shaker
4. Store at -20°C
GLYCEROL STOCK SOLUTION PROTOCOL
1. Take 500µL of a previously prepared culture and place into a screw-top eppendorf tube
2. Add 500µL of Glycerol solution (V.V 50% to make a 25% concentration
3. Place in -20°C storage
QIAGEN MINIPREP PROTOCOL
1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, solution turns blue.
3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, solution turns colorless.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
5. Apply the supernatant (from step 4) to the QIAprep spin column by decanting or pipetting.
6. Centrifuge for 30–60 s. Discard the flow-through.
7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through. This step is only required when using endA+ or other bacteria strains with high nuclease activity or carbohydrate content (see QIAprep Miniprep Handbookfor more details)
8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
10. To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 µl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
GEL ELECTROPHORESIS PROTOCOL
1. Measure 0.6g of agarose and add to 60 mL TAE Buffer
2. Mix throroughly
3. Heat until solution becomes clear
4. Allow to cool, then add 30µl of 1mg per ml Ethidium Bromide
5. Pour solution into gel tray with comb in place
6. Allow to set
7. Place in electrophoresis tank and submerge in TAE buffer
8. Pipette 10 µl of ladder into left most well
9. Perform serial dilution of sample
10. Add 9 µl of sample to 1 µl of loading dye and place in wells, noting position of each sample
11. Run gel at 90V for 30 minutes
2. Visualise using UV light
PCR PROTOCOL
1. Program desired cycle into PCR machine. Cycle used was as follows:
94°C - Initial Denaturation - 120 seconds
94°C - 30 seconds
58-68°C - Gradient Cycle - 30 seconds
68°C - Final Extension - 150 seconds
68°C - 300 seconds
4°C - Hold
Second, third and fourth stages were repeated for 30 cycles.
2. Three serial dilutions were conducted, yielding concentrations of 1:10, 1:100, and 1:1000. This was carried out in order to find the optimum concentration of template DNA to use, as we didn't know the concentration yielded from the Miniprep Protocol used to extract the plasmid. We conducted a gradient cycle because even though we knew the annealing temperatures of the primers we'd designed, we wanted to make sure we had the optimum temperature range.
PEG Transformation
1. Add 9ml of 8% mannitol to a petri dish.
2. Using a spatula, put 7 day old moss from 2-3 PPNH4 plates in the petri dish containing the mannitol.
3. Add 3ml of 2% driselase to the petri dish.
4. Incubate the petri dish at room temperature with gentle shaking for 1 hour.
5. Filter the protoplast suspension through a 100µm mesh.
6. Spin the filtered suspension at 250g for 5 minutes.Remove the supernatant.
7. Resuspend the protoplasts very gently with 500µl of 8%mannitol.
8. Add 9.5ml 8% mannitol in the culture tube. Make sure the protoplasts are fully suspended.
9. Repeat the filtration and re-suspension steps (6,7,8) two more times.
10. Take 10µl of the protoplast solution and count the protoplasts using a haemocytometer.
11. Multiply the number of protoplasts in as 16 square area by 10,000 to obtain the amount of protoplastspts ml.
12. Spin the protoplast solution at 250g for 5 minutes. Remove the supernatant.
13. Re-suspend protoplasts in MMg solution at the concentration 1.6 million protoplasts/ml./p>
14. Incubate the protoplast suspension at room temperature for 20 minutes.
15. Add 600µl of protoplast suspension into a culture tube containing 60µg DNA. Swirl the tube gently.
16. Add 700µl of PEG/Ca solution into the protoplast/DNA mixture. Swirl the tube gently until all the mixture homogeneous.
17. Incubate the mixture at room temperature for 30 minutes.
18. During this waiting period cover PRM-B plates with 80mm cellophane discs. Allow the cellophane to hydrate on the plate surface for at least 5 minutes.
19. With a spatula remove any air bubbles trapped between the cellophane and the plate.
20. Dilute the mixture with 3ml of W5 solution.
21. Spin the mixture at 250g for 5 minutes. Remove the supernatant.
22. Re-suspend protoplasts in melted 2ml of PRM-T. Plate 1ml of re-suspended protoplasts per PRM-B plate covered by cellophane. Wrap the plates with micropore tape and keep in a 25°C growth chamber.
Growth chamber is set to 16hrs light 8hrs dark cycle.
Move the cellophane onto a fresh selection plate 4 days after transformation.
