Complementation in Arabidopsis
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Complementation is performed to rescue the phenotype of a mutant plant with the cloned wildtype gene. This can prove that the gene cloned is in fact the gene that was mutated in the mutant plant.
I. Cloning wildtype gene into the plant transformation vector:
1. Design primers for region to be amplified.
~undefinedDesign_the_primers_with_a_restriction_site_at_the_5~9_end_of_either_strand_so_that_the_cloned_region_can_be_ligatedinto_a_vector_with_the_same_site._Also_include_4~F5_extra_bases_after_the_restriction_site._To_summarize~I_the_primers_should_have_20_bases_specific_to_the_region_to_be_amplified~E_6_bases_for_the_restriction_site~E_and_4_extra_bases_for_a_total_length_of_30_bases_per_primer.~Kbr_~H~M~2~1~0~Kbr_~H~M~2~1~02._Run_PCR_~Aon_fewer_cycles_20_to_25~B_with_wildtype_DNA_according_to_the_recipe_below~I_~ARun_4_replicates~HDNA_sample~B~Kbr_~H~M~2~1~0
|
Template DNA
Forward Primer (20µM) Reverse Primer (20µM) 10X PCR Buffer dNTPs (2.5 mM each) ddH20 +Taq (ExTaq~undefined TOTAL |
1µL
0.5µL 0.5µL 5µL 4µL 40µL 1µL 52µL |
3. Combine reactions of same template and tun 5µL on an agarose gel to verify the PCR successfully amplified the desired region.
4. Purify the PCR products. If there is a single band, use the PCR Purification protocol outlined in the Qiagen Quickspin kit. If there are multiple bands, follow the Gel Extraction protocol (also outlined in the Qiagen Quickspin kit) to purify the correct band. Remember to elute the DNA fragment with 50µL of ddH20 prewarmed to 65°C.
5. Digest the purified DNA and vector-to-be-used with the restriction enzyme that will cut the ends of the cloned region and vector. The vector should also contain antibiotic resistance for selecting transformed plants (e.g. we use Kanamycin). Digest for ~6 hours and use following recipe:
|
DNA 10X Buffer ddH20 Emzyme TOTAL |
PCR Product
20µL 10µL 65µL 5µL 100µL |
Vector
10µL 10µL 75µL 5µL 100µL |
6. Purify the vector and PCR product from the digestion using the Qiagen Quickspin kit. Elute in 30µL of ddH2O, prewarmed to 65°C.
7. Remove 5' phosphate from the digested vector (to prevent re-annealing) by incubating with SAP (Shrimp Alkaline Phosphatase) for 2 hours at 37°C, then at 70°C for 20 minutes to inactivate SAP enzyme. Use the recipe below:
|
Purified, Digested Vector
10x SAP Buffer SAP ddH2O TOTAL |
30µL
10µL 5µL 55µL 100µL |
8. Next, the vector (which can not re-ligate) and cloned region (now with "sticky" ends) need to be ligated together. Prepare the ligation reactions according to the recipes below:
|
Vector PCR Product T4 Ligase Buffer Ligase ddH2O TOTAL |
CONTROL
4µL 0µL 1µL 1µL 4µL 10µL |
LIGATION
7µL 1µL 1µL 1µL ----- 10µL |
9. Run the ligation reactions at 16°C overnight.
10. Transform E. Coli chemi-competent TOP10 cells with the construct:
a. Add 5µL reaction to 1 tube of cells. Stir mixture with pipette tip; DO NOT PIPETTE.
b. Keep on ice for 15 minutes.
c. Heat shock mixture at 42°C for 30 second.
d. Remove tube(s) from 42°C water bath and return to ice for 1-2 minutes then add 250µL SOC media.
e. Shake for 1 hour at 37&ded;C to allow cells to revcover. Put multiple tubes into a flask and then put in floor shaker.
11. Plate 2 plates each for the control and ligation reactions. The LB should contain Kanamycin since the construct encodes resistance. For the control reaction, one plate should be 20µL of the recovered cells and the other should be 100µL of the recovered cells. Plate the same amounts for the ligation reaction.
12. After the plates are allowed to grow overniht at 37°C, analyze several colonies on the ligation reaction plates to see if the construct is there via colony PCR. For the control reaction plates, it is only necessary to analyze 3 colonies. NOTE: Use primers specific for the gene of choice in Step 1.
13. Create 'back-up' colony plates for each colony screened. Draw a grid on the back of a LB plate. Touch the colony with a pipette tip and streak it across the media within a single square in the grid. Write the colony number in the space where the pipette tip has been streaked. Do this for all colonies screened in previous step. Grow the plates overnight at 37°C.
14. Using the colony PCR results, select the colonies with tbrightest specific PCR product. Use the 'back-up' plates to retrieve a clone of that individual and inoculate liquid media cultures (with Kanamycin). Also select one empty colony to grow. Grow the cultures in a shaker overnight at 37°C.
15. Plasmid purifiy each culture following the Qiagen protiocol for plasmid prep.
16. Select a restriction enzyme that will cut your construct in a manner that the orientation of the insert will be clear. Digest the prep'd plasmids and analyze the individuals on an agarose gel.
17. Once the orientation of the constructs has been confirmed, select an individual with a sense insertion and sequence the cloned gene to assure no mutations were obtained throughout the PCR process.
II. Introduction of complementation construct into Agrobacterium:
18. If there are no mutations within the cloned region (from step 1), transform Agrobacterium with the plasmid of choice following this recipe:
1µL plasmid with sense insertion
1µL 'helper' plasmid, we use pSOUP (with Carbenicillin resistance.)
1 tube Agro GV3101 Electro-competent cells (GV3101 cells are resistant to Rifampicin and Gentamycin.)
19. Shock the mixture at 25µF, 400 Ohms, and 2.5kV.
20. Add 1 mL SOC to the mixture and shake the cells at 28°C for 2 hours.
21. From the recovered Agro cells, plate 10µL and 100µL on LB media containing antibiotics (Rifampicin and Gentamycin for the Agro, Carbenicillin for pSOUP helper plasmid, and Kanamycin for the construct. Do you have the concentrations of the antibiotics?) to select colonies that received the plasmid. Allow them to grow for 2 days at 28°C.
22. Select 10 random colonies, number them and perform Colony PCR on them. Visualize the results of the PCR on an agarose gel. NOTE: Use primers specific for the gene of choice in Step 1.
23. Choose 3 colonies that are positive for the construct and inoculate 2mL LB cultures with antibiotics (Rifampcin , Gentamycin , Carbenicillin , and Kanamycin , or selection marker of your choice)for each colony. Allow cultures to grow for 48 hours at 28°C in a floor shaker.
III. Transformation of plants.
24. Once the cultures have grown up, take 1/2 mL from each culture and add that to 500 mL of LB liquid media with all antibiotics used in previous step. Grow the cultures in a floor shaker at 28°C overnight.
25. Transfer the contents of the cultures to centrifuge bottles and spin them down. NOTE: When using a centrifuge, balance the volume in the bottles to prevent the rotor from warping.
26. Decant and pippette off the supernatant.
27. Resuspend the pellets in 500mL of Plant Transformation Medium by swirling the medium in the bottles or letting them sit. DO NOT pipette, shake, or vortex to resuspend. Use the following recipe for Plant Transformation Medium:
|
Sucrose MS Salt MES Benzylaminopurine MS Vitamin SILWET ddH2O |
1L
50 grams 2.2 grams 0.5 grams 10µL 1mL 200µL 1 Liter |
2L
100 grams 4.4 grams 1 gram 20µL 2mL 400µL 2 Liter |
29. There are two ways to transform Arabidopsis plants. 1) Dip the plant in the Plant Transformation Medium. Simply take the enture pot, use one hand to prevent soil from falling out of the pot, and submerge the entire plant in a container filled with the transformed Agro Plant Transformation Medium. Leave the plant submerged for approximately 1 minute. Let the plants dry for a few hours before returning them to the growth chamber. Dip the plants every 3 days, do this twice more so that the plants are dipped a total of 3 times. 2) Spray the plants with the Plant Transformation Medium. Fill a bottle with the transformed Agro Plant Transformation Medium and attach a spray nozzle. Spray the plants copiously and return them to the growth chamber. Spray the plants every 3 days; do this twice more so that the plants are sprated a total of 3 times.
30. Let the transformed plants set seeds. Avoid over watering as mold will grow due to the sugar-rich transformation medium. Collect seeds, dessicate and plate them on appropriate selection medium to obtain transformants. <center> <p> </p> </center>
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