| Complementation in Arabidopsis | 点击： 作者：51protocol收集 来源： 时间： 2007-03-23 本站论坛
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|  | | 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
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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, Carbenicillinfor 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.
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