Difference between revisions of "Team:Michigan/Experiments"
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1. Make modifications to the plasmids we’re ordering and the one from Dr.Zhang <br /> | 1. Make modifications to the plasmids we’re ordering and the one from Dr.Zhang <br /> | ||
a. Use PCR to amplify the plasmids<br /> | a. Use PCR to amplify the plasmids<br /> | ||
| − | b. Point mutagenesis with specific primers to introduce digestion sites (to Dr.Zhang’s Plasmid) | + | b. Point mutagenesis with specific primers to introduce digestion sites (to Dr.Zhang’s Plasmid)<br /> |
| − | c. Transform in DH5α and plate to isolate colonies | + | c. Transform in DH5α and plate to isolate colonies<br /> |
| − | i. Check to ensure that our point mutation is correct either by Sanger Sequencing or colony PCR with the mutated primers | + | i. Check to ensure that our point mutation is correct either by Sanger Sequencing or colony PCR with the mutated primers<br /> |
| − | d. Digest at the newly introduced sites to cut out the portion of Dr.Zhang’s plasmid that we need | + | d. Digest at the newly introduced sites to cut out the portion of Dr.Zhang’s plasmid that we need <br /> |
| − | e. Ligate this insert into one of the empty backbones from iGEM | + | e. Ligate this insert into one of the empty backbones from iGEM<br /> |
| − | f. Clone the SaCas9 that was order into the other empty backbone from iGEM | + | f. Clone the SaCas9 that was order into the other empty backbone from iGEM<br /> |
| − | 2. Make point mutations to the WT-Cas9 plasmids to disable nuclease activity and make them dCas9 | + | 2. Make point mutations to the WT-Cas9 plasmids to disable nuclease activity and make them dCas9<br /> |
| − | a. Use primers introduce these point mutations in each of the plasmids | + | a. Use primers introduce these point mutations in each of the plasmids <br /> |
| − | b. Confirm the mutations by transformation into DH5α to separate colonies and submit to purified sample to Sanger Sequencing | + | b. Confirm the mutations by transformation into DH5α to separate colonies and submit to purified sample to Sanger Sequencing<br /> |
| − | 3. Transform the testing plasmid into BL21 Competent E. coli and use corresponding selection antibiotic to confirm | + | 3. Transform the testing plasmid into BL21 Competent E. coli and use corresponding selection antibiotic to confirm<br /> |
<p> | <p> | ||
Revision as of 00:33, 29 June 2018
Experiments
Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.
1. Make modifications to the plasmids we’re ordering and the one from Dr.Zhanga. Use PCR to amplify the plasmids
b. Point mutagenesis with specific primers to introduce digestion sites (to Dr.Zhang’s Plasmid)
c. Transform in DH5α and plate to isolate colonies
i. Check to ensure that our point mutation is correct either by Sanger Sequencing or colony PCR with the mutated primers
d. Digest at the newly introduced sites to cut out the portion of Dr.Zhang’s plasmid that we need
e. Ligate this insert into one of the empty backbones from iGEM
f. Clone the SaCas9 that was order into the other empty backbone from iGEM
2. Make point mutations to the WT-Cas9 plasmids to disable nuclease activity and make them dCas9
a. Use primers introduce these point mutations in each of the plasmids
b. Confirm the mutations by transformation into DH5α to separate colonies and submit to purified sample to Sanger Sequencing
3. Transform the testing plasmid into BL21 Competent E. coli and use corresponding selection antibiotic to confirm
Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.
What should this page contain?
- Protocols
- Experiments
- Documentation of the development of your project