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</div> <br /><br /><br /><br /><br /><br /> | </div> <br /><br /><br /><br /><br /><br /> | ||
− | <section><h3>1. Cloning of module 1:</h3><h4> | + | <section><h3>1. Cloning of module 1:</h3><h4>Construct 1--> Ptet - RBS1 - T7 RNA polymerase - terminator 1 - terminator 2. |
Two PCR reactions were performed. | Two PCR reactions were performed. | ||
− | First PCR reaction | + | First PCR reaction was performed with primer 1 and 2 using the template R0040 to amplify the Ptet. The size of PCR product is 77 bp. |
− | Second PCR reaction | + | Second PCR reaction was performed with primer 3 and 4 using the template K1450004 to amplify the RBS1 - T7 RNA polymerase - terminator 1 - terminator 2. The size of this PCR product is 2338 bp. |
− | + | Thirdly, SOE PCR was performed using the primers 1 and 4 (for PCR conditions and reaction mixture, refer notebook). | |
</h4> | </h4> | ||
<center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/8/8c/T--IISER-Mohali-INDIA--CS2.png" alt="Flow chart"></td></tr></table></center> | <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/8/8c/T--IISER-Mohali-INDIA--CS2.png" alt="Flow chart"></td></tr></table></center> | ||
− | <h4>The final PCR product was | + | <h4>The final PCR product was attempted to be cloned at AatII and ApaI sites of pZS21MCS. However, we did not get positive results for cloning, resulting in failure to clone the construct 1. |
− | + | Similarly, cloning of construct 2 (PT7 - RBS2 - Chromoprotein II - RBS3 - tetR - terminator3), three PCR reactions were performed.</h4> | |
− | Similarly, | + | |
<center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/b/b3/T--IISER-Mohali-INDIA--CS3.png" alt="Flow chart"></td></tr></table></center> | <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/b/b3/T--IISER-Mohali-INDIA--CS3.png" alt="Flow chart"></td></tr></table></center> | ||
− | <h4>First PCR reaction | + | <h4>First PCR reaction was performed with primer 5 and 6 using the template K1343022 to amplify the PT7-RBS2. The size of PCR product is 132 bp. |
− | Second PCR reaction | + | Second PCR reaction was performed with primer 7 and 8 using the template K1033910 to amplify the chromoprotein II. The size of PCR product is 759 bp. |
Third PCR was performed using the primers 9 and 10 (for PCR conditions and reaction mixture, refer notebook). | Third PCR was performed using the primers 9 and 10 (for PCR conditions and reaction mixture, refer notebook). | ||
− | Then SOE1 PCR was performed using primer 5 and 8 to get 891 bp | + | Then, SOE1 PCR was performed using primer 5 and 8 to get 891 bp amplified fragment. |
− | Then SOE2 PCR was performed using using primer 5 | + | Then, SOE2 PCR was performed using using primer 5, 10 and the template SOE1 (891bp) fragment to amplify 1783 bp fragment. |
− | This SOE2 PCR | + | This SOE2 PCR fragment was attempted to clone in low copy number plasmid pZS21MCS, at SalI and BamHI site. But due to problems with digestion of vector, we were not able to clone in pZS21MCS plasmid. |
Therefore, construct 2 was finally cloned at SalI and BamHI site of pRC10 (modified ptrc99a) which is intermediate copy number plasmid. | Therefore, construct 2 was finally cloned at SalI and BamHI site of pRC10 (modified ptrc99a) which is intermediate copy number plasmid. | ||
The <i>E. coli</i> transformants were screened for positive clones of construct 2 by restriction digestion and obtained positive clone 9 and shown in agraose gel below. | The <i>E. coli</i> transformants were screened for positive clones of construct 2 by restriction digestion and obtained positive clone 9 and shown in agraose gel below. |
Revision as of 20:30, 1 November 2017