IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/28: Difference between revisions
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|style="background-color: #EEE"|[[Image: | |style="background-color: #EEE"|[[Image:TeamLogo.jpg|350px]]<span style="font-size:19px;"> UNAM-Genomics-Mexico team</span> | ||
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | |style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | ||
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Revision as of 14:25, 9 October 2010
UNAM-Genomics-Mexico team | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||
Working on cI inverter construction and fusion to pSB3K3 backbone. LovTAP repressor activity reporter systemOnce the plasmids harboring the parts showed in the next table were correctly isolated, I am going to digest P0451(RBS+cI repressor) with SpeI and PstI, this will be ligated to the Part K098991 (cI regulated promoter+RBS+GFP) digested with XbaI and PstI in order to fuse them to construct the whole cI inverter. Then it will be ligated to trpL and J23101 promoters.
Plasmid digested: Plasmid harboring P0451 isolated from colony 6.
Note: Inactivate the enzymes at 80°C during 20 min. Repeated experiment:Working on cI inverter construction and fusion to pSB1C3 backbone. LovTAP repressor activity:reporter systemDue to the first attempt to ligate K098991 (cI regulated promoter+RBS+GFP) and P0451(RBS+cI repressor) inside plasmid pSB1C3 failed, I am going to repeat experiment. Ligation Procedure: P0451 + K098991 to plasmid pSB1C31. Prepare the ligation mixture taking into account the quantity of the DNA insertS -K098991 and P0451- and the receiver DNA -plasmid pSB1C3-. This can be check in the following gel. Quantity loaded 3μL.
2. Incubate the sample at 16°C overnight. 3. Transform the cells. Click here for the protocol. 4. Culture the cells in the proper selective medium, in our case LB + Kanamycin. 5. Incubate the petri dishes at 37°C overnight. 6. Re-culture the resultant colonies in the proper selective medium; incubate them at 37°C overnight. 7. Analyze the colonies with Colony PCR to confirm that they contain the correct ligation The primers that I am using are: Forward (5'->3'): Preffix primer. Reverse:(5'->3'): Suffix primer. These primers would amplify the whole cI inverter, if the ligation was correctly done. Repeated experiment:Working on J23102 promoter: Ligation to P0451/Lumazine/LuxYDue to the first attempt to ligate P0451, Lumazine and LuxY to promoter J23102 failed, I am going to repeat the experiment. Ligation Procedure:Promoter J23102 with P0451 (RBS+cI repressor)/Lumazine and LuxY1. Prepare the ligation mixture taking into account the quantity of the DNA insert -P0451 (RBS+cI repressor),LuxY and Lumazine- and the receiver DNA -plasmid harboring the promoter J23102-. This can be check in the following gel.
2. Incubate the sample at 16°C overnight. 3. Transform the cells. Click here for the protocol. 4. Culture the cells in the proper selective medium, in our case LB + Kanamycin. 5. Incubate the petri dishes at 37°C overnight. 6. Re-culture the resultant colonies in the proper selective medium; incubate them at 37°C overnight. 7. Analyze the colonies with Colony PCR to confirm that they contain the correct ligation The primers that I am using are: Forward (5'->3'): Preffix primer. Reverse:(5'->3'): Suffix primer. These primers would amplify LuxY, Lumazine and P0451:RBS+cI repressor plus promoter J23101, if the ligation was correctly done.
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