The Preuss Lab,The Division of Biological Sciences,The University of Chicago
http://preuss.bsd.uchicago.edu/protocols/complementation.html
(per Adam 08/12/04)
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.
**Design the primers with a restriction site at the 5' end of either strand so that the cloned region can be ligatedinto a vector with the same site. Also include 4-5 extra bases after the restriction site. To summarize: the primers should have 20 bases specific to the region to be amplified, 6 bases for the restriction site, and 4 extra bases for a total length of 30 bases per primer.
2. Run PCR (on fewer cycles 20 to 25) with wildtype DNA according to the recipe below: (Run 4 replicates/DNA sample)
|
Template DNA
Forward Primer (20µM)
Reverse Primer (20µM)
10X PCR Buffer
dNTPs (2.5 mM each)
ddH20
Taq (ExTaq)*
TOTAL |
1µL
0.5µL
0.5µL
5µL
4µL
40µL
1µL
52µL |
*Add after 4 minutes of 95°C for Hot Start 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 |
| |
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