Difference between revisions of "Part:BBa K1955005"
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The pSB1C3-HA-3’UTR and pSB1C3-OVA-3’UTR were digested with EcoRI and XbaI, while the pSB1C3-5’UTR was digested with EcoRI and SpeI. The pSB1C3-HA-3’UTR, pSB1C3-OVA-3’UTR and 5’UTR were purified by gel extraction, and ligated together. After the transformation step, we used colony PCR to check the correctness of the plasmid. The results showed that the approximately 4100 bp long 5’HYG-HA-3’UTR (1446 bp +1700 bp + 774 bp) and 4500 bp 5’HYG-HA-3’UTR (1446 bp + 2098 bp+ 774 bp) could be amplified from the plasmid, meaning that the pSB1C3-HA-3’UTR, pSB1C3-OVA-3’UTR were finished in the step. In order to transfect the plasmid into leishmania by electroporation, we amplified the plasmid in 200 ml LB broth, and purified the DNA by midiprep. | The pSB1C3-HA-3’UTR and pSB1C3-OVA-3’UTR were digested with EcoRI and XbaI, while the pSB1C3-5’UTR was digested with EcoRI and SpeI. The pSB1C3-HA-3’UTR, pSB1C3-OVA-3’UTR and 5’UTR were purified by gel extraction, and ligated together. After the transformation step, we used colony PCR to check the correctness of the plasmid. The results showed that the approximately 4100 bp long 5’HYG-HA-3’UTR (1446 bp +1700 bp + 774 bp) and 4500 bp 5’HYG-HA-3’UTR (1446 bp + 2098 bp+ 774 bp) could be amplified from the plasmid, meaning that the pSB1C3-HA-3’UTR, pSB1C3-OVA-3’UTR were finished in the step. In order to transfect the plasmid into leishmania by electroporation, we amplified the plasmid in 200 ml LB broth, and purified the DNA by midiprep. | ||
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| − | <img src="https://static.igem.org/mediawiki/2016/1/1c/CGU_Taiwan--bio7.jpg" width=350px height= | + | <img src="https://static.igem.org/mediawiki/2016/1/1c/CGU_Taiwan--bio7.jpg" width=350px height=250px style="border:2px black solid;border-radius:8px;"></img><br><br> |
pSB1C3-HA-3’UTR, pSB1C3-OVA-3’UTR checked by colony PCR<br><br> | pSB1C3-HA-3’UTR, pSB1C3-OVA-3’UTR checked by colony PCR<br><br> | ||
The pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR were transformed and the colonies were picked to perform colony PCR. The PCR reaction was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis. The 4100 bp 5’HYG-HA-3’UTR and 4500 bp 5’HYG-OVA-3’UTR were amplified from pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR. | The pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR were transformed and the colonies were picked to perform colony PCR. The PCR reaction was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis. The 4100 bp 5’HYG-HA-3’UTR and 4500 bp 5’HYG-OVA-3’UTR were amplified from pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR. | ||
Revision as of 09:06, 20 October 2016
pSB1C3-5'HYG-HA-3'UTR
The complete coding sequence of Ovalbumin (OVA) acquired from GenBank (Accession# AH002466.2) . OVA has been commonly used as the antigen for testing the efficiency of antibody response and T cell activation in previous immunology experiments, thus is a easy to use antigen.
(1) The construction of pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR:
The pSB1C3-HA-3’UTR and pSB1C3-OVA-3’UTR were digested with EcoRI and XbaI, while the pSB1C3-5’UTR was digested with EcoRI and SpeI. The pSB1C3-HA-3’UTR, pSB1C3-OVA-3’UTR and 5’UTR were purified by gel extraction, and ligated together. After the transformation step, we used colony PCR to check the correctness of the plasmid. The results showed that the approximately 4100 bp long 5’HYG-HA-3’UTR (1446 bp +1700 bp + 774 bp) and 4500 bp 5’HYG-HA-3’UTR (1446 bp + 2098 bp+ 774 bp) could be amplified from the plasmid, meaning that the pSB1C3-HA-3’UTR, pSB1C3-OVA-3’UTR were finished in the step. In order to transfect the plasmid into leishmania by electroporation, we amplified the plasmid in 200 ml LB broth, and purified the DNA by midiprep.
pSB1C3-HA-3’UTR, pSB1C3-OVA-3’UTR checked by colony PCR
The pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR were transformed and the colonies were picked to perform colony PCR. The PCR reaction was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis. The 4100 bp 5’HYG-HA-3’UTR and 4500 bp 5’HYG-OVA-3’UTR were amplified from pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1458
Illegal BamHI site found at 3164 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 3094