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Team:UEA-JIC Norwich/Methods
From 2011.igem.org
High Efficiency Transformation Protocol
Culture Preparation Protocol
Glycerol Stock Solution Protocol
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 Protocol
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.
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.
Algae Transformation Method using Glass Beads
1. Transfer 50ml of algae culture into four 50ml screw top tubes.
2. Place all four tubes in the eppendorf centrifuge at 800rpm for 9 minutes at 20-22°C.
3. Immediately pour contents of tubes into glass beaker and keep tubes upside down so no unwanted suspension mixes with the pellet.
4. For all tubes resuspend cells in 750µl of TAP media.
5. Wash pre prepared beads in ethanol removing excess gently.
6. Wash beads approximately 2-3 times in autoclaved distilled water.
7. Place approximately half the beads in a 1.5ml eppendorf tube for the control.
8. Resuspend both tubes with 200µL of distilled water.
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Algae Transformation Method using Electroporation
1. Transfer 50ml of algae culture into four 50ml screw top tubes.
2. Place all four tubes in the eppendorf centrifuge at 800rpm for 9 minutes at 20-22°C.
3. Immediately pour contents of tubes into glass beaker and keep tubes upside down so no unwanted suspension mixes with the pellet.
4. For all tubes resuspend cells in 250µl of TAP and sucrose media.
5. Add linearised G-Luc plasmid (5µL)to the suspension and place on ice.
6. Transfer 250µL of algae suspension into 1mL cuvette.
7. Subject cuvettes to electroporation at 2200v/ cm and place back on ice.
8. Add 500µL of starch solution to each cuvette and aspirate gently.
9. Add 750µL of each sample onto an LB plate.
10.Parafilm the lid and plate to seal and place in a light incubator.
Starch Preparation
1. Wash starch powder with 10mL distilled water and resuspend.
2. Centrifuge for five minutes at 4000rpm.
3. Pour off supernatant.
4. Resuspend with 500µL Ethanol.
5. Centrifuge for five minutes at 4000rpm.
6. Resuspend in 500µL TAP.
7. Centrifuge for five minutes at 4000rpm, pour off supernatant, and repeat once.
8. Resuspend in 500µL TAP with sucrose.
