Team:WashU/Week5
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We have also decided to use PCR to amplify our DNA, as we are getting extremely low yields from our gel extractions. Thus, we designed the primers today. | We have also decided to use PCR to amplify our DNA, as we are getting extremely low yields from our gel extractions. Thus, we designed the primers today. | ||
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The constructs were all cloned in GC5 competent cells from sigma using the transformation protocol. The J23119 cut with SpeI and PstI constructs were plated onto Amp. The Kan and C plasmid constructs were plated onto their respective resistance plate. The plates were incubated overnight at 37°C. | The constructs were all cloned in GC5 competent cells from sigma using the transformation protocol. The J23119 cut with SpeI and PstI constructs were plated onto Amp. The Kan and C plasmid constructs were plated onto their respective resistance plate. The plates were incubated overnight at 37°C. | ||
- | Today, we began a culture of <i>E. coli</i> transformed with plasmid PSL2131. | + | Today, we began a culture of <i>E. coli</i> transformed with plasmid PSL2131. |
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==Saffron in a Kan== | ==Saffron in a Kan== | ||
- | We finally received our gene from DNA 2.0!! We streaked out some of the gene, still in its plasmid | + | We finally received our gene from DNA 2.0!! We streaked out some of the gene, still in its plasmid onto plates with ampicillin. |
- | In addition, we transformed <i>Synechocystis</i> with the plasmid PSL2131 obtained from one of our mentors, Burt. | + | In addition, we transformed <i>Synechocystis</i> with the plasmid PSL2131 obtained from one of our mentors, Burt. |
- | We picked the <i>E. coli</i> colonies transformed with plasmid PSL2131 today. | + | We picked the <i>E. coli</i> colonies transformed with plasmid PSL2131 today. We miniprepped these colonies and then put them into 6803. |
- | We have started a third wild type liquid culture of <i>Synechocystis</i>. | + | We have started a third wild type liquid culture of <i>Synechocystis</i>. |
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==YLC== | ==YLC== | ||
- | In the interest of time, we will use existing biobrick constructs with the necessary fluorescent proteins to show to the YLC students, and then we will complete our own constructs at a later date. Thus, today, we plated several biobrick constructs | + | In the interest of time, we will use existing biobrick constructs with the necessary fluorescent proteins to show to the YLC students, and then we will complete our own constructs at a later date. Thus, today, we plated several biobrick constructs (RFP: I13521, |
+ | GFP: I13522, | ||
+ | YFP: 13604, and | ||
+ | CFP: S03475) and will pick colonies to prep. | ||
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==Saffron in a Kan== | ==Saffron in a Kan== | ||
We continued to work with our gene construct today. <br> | We continued to work with our gene construct today. <br> | ||
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- | + | We made glycerol stocks of GFP, RFP, plasmid 2131, and then three of our construct, CS42S. | |
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+ | We performed a miniprep to extract the DNA PSL2131.<br> | ||
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+ | We performed two digestions - see the gel below for results.<br> | ||
[[File:629122gel.jpg]] | [[File:629122gel.jpg]] | ||
+ | [[File:6292012322.jpg]] | ||
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+ | <u>Saturday, June 30</u> | ||
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+ | ==Several Small Projects== | ||
+ | We miniprepped the potential constructs. We will need to digest them to check if they worked. They will hopefully be the plasmid that we want to place into Synechocystis. | ||
+ | <br> | ||
+ | More Amp and Kan LB plates were made. | ||
+ | <br> | ||
+ | Four more glycerol stocks of the construct that we ordered were made. 2 mL of the remaining cells were miniprepped. The rest (.25 mL) were used to continue the line of cells in liquid culture with 3 mL of LB+Amp. | ||
+ | <br> | ||
+ | Two glycerol stocks one of an RFP plasmid and one of our functioning GFP were made and frozen at -80°C. (For the glycerol stocks done today, no dry ice was used to flash freeze as a the stock room is closed on Saturdays.) | ||
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Latest revision as of 16:43, 17 August 2012