Team:WashU/Week6
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==Digest To Check Construct== | ==Digest To Check Construct== | ||
The minipreped constructs from Saturday were digested with XbaI and PstI to check if they contained the desired plasmid and insert. | The minipreped constructs from Saturday were digested with XbaI and PstI to check if they contained the desired plasmid and insert. | ||
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- | + | A 1% agarose gel was made to check the digests of the ligation. | |
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- | A 1% agarose gel was made to check the digests of the ligation. | + | |
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The ligations were deemed unsuccessful, and most were likely the plasmid backbone with the small self-ligated addition filling in the cut. We will re-digest the insert and plasmid and gel purify a large quantity of DNA to reduce the risk of self-ligation. | The ligations were deemed unsuccessful, and most were likely the plasmid backbone with the small self-ligated addition filling in the cut. We will re-digest the insert and plasmid and gel purify a large quantity of DNA to reduce the risk of self-ligation. | ||
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==YLC Color Constructs== | ==YLC Color Constructs== | ||
- | The plates all have a good amount of colonies on them. | + | The plates all have a good amount of colonies on them. |
The RFP and GFP are glowing well after about 14 hours of incubation. They are now sitting and room temperature while the CFP and YFP plates are placed back in the incubator to further develop. | The RFP and GFP are glowing well after about 14 hours of incubation. They are now sitting and room temperature while the CFP and YFP plates are placed back in the incubator to further develop. | ||
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==Saffron Constructs Preparation== | ==Saffron Constructs Preparation== | ||
Today, the Kan-plasmid which we are using as the construct vector and the CS42s construct to produce Saffron from Zeaxanthin were both digested with XbaI and SpeI. These digests were then run on a gel to prepare for gel purification. The gel from yesterday was not a clear gel result so this gel was a .35% gel to try to further see separation of bands.<br> | Today, the Kan-plasmid which we are using as the construct vector and the CS42s construct to produce Saffron from Zeaxanthin were both digested with XbaI and SpeI. These digests were then run on a gel to prepare for gel purification. The gel from yesterday was not a clear gel result so this gel was a .35% gel to try to further see separation of bands.<br> | ||
- | + | In the gel below, P refers to the plasmid PSL2131, and C and CIII refer to two different colonies of our CS42S construct. | |
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<img src="https://static.igem.org/mediawiki/2012/c/ce/Successjuly3.tif" width="965px"> | <img src="https://static.igem.org/mediawiki/2012/c/ce/Successjuly3.tif" width="965px"> | ||
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+ | Afterwards, gel purification was done on the plasmid and constructs. However, due to some error on our part, no DNA was recovered, as shown by the nanodrop. | ||
==Glycerol Stock Check== | ==Glycerol Stock Check== | ||
The stock that was grown in LB+Amp overnight was cloudy suggesting cell growth. A glycerol stock that was made on Saturday of a GFP will be checked today by plating it on an LB+Amp plate. This test will be more conclusive in showing the ability of the glycerol stock to not only produce growing bacteria but also maintain plasmid at -80°C. | The stock that was grown in LB+Amp overnight was cloudy suggesting cell growth. A glycerol stock that was made on Saturday of a GFP will be checked today by plating it on an LB+Amp plate. This test will be more conclusive in showing the ability of the glycerol stock to not only produce growing bacteria but also maintain plasmid at -80°C. | ||
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+ | ==PCR== | ||
+ | We ran a gel on the rest of yesterday's PCR. The gel, shown below, suggests the presence of primer dimers indicating that our PCR failed. After troubleshooting, we will attempt to get the colony PCR to work anew. (In the gel, A and K refer to Ampicillin and Kanamycin plasmids, Z refers to the ZCD gene, U refers to UGTCS2, and C refers to CrtZ.) <br> | ||
+ | https://static.igem.org/mediawiki/2012/0/0e/PCR-July6.jpg | ||
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+ | Troubleshoot and redo failed PCRs | ||
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+ | ==6803== | ||
+ | Because our first attempt at transforming 6803 with a kanamycin resistant vector did not succeed due to the plates dying out, we attempted to carry out the procedure again using our new incubator. We plated a total of four BG-11 plates, giving us the ability to do a 2x2 test of plate conditions. We chose to test the effect of mixotrophic media (with and without glucose) and amount of transformed cells plated (50ul vs 200ul) on the transformation efficacy. | ||
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+ | <u>Saturday, July 8</u> | ||
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+ | First, we reinnoculated the ligation cultures.<br> | ||
+ | Then, we maxipreped PsbA2 and used the nanodrop to see how much DNA resulted from the prep. | ||
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+ | Next, we digested the ligations, miniprepped and digested them, made a gel, and ran the gel. [PICTURE] | ||
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+ | In addition, we started a 20 degree culture of <i>E. coli</i> with the Z construct and our construct CS42S. | ||
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+ | We miniprepped the <i>E. coli</i>which had taken up double plasmids (Z and C plasmids). We digested these and then ran them on a gel. [PICTURE] | ||
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+ | Finally, we replated ligation 2, our most successful ligation of plasmid PSL2131 and construct CS42S. | ||
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+ | <td2><a href="/Team:WashU/Week5"><img src ="http://aux4.iconpedia.net/uploads/937908040.png" width=82px height=82px></a></td2> | ||
+ | <td2><a href="/Team:WashU/Week7"><img src ="http://aux.iconpedia.net/uploads/1782679686.png" width=82px height=82px></a></td2> | ||
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[https://2012.igem.org/Team:WashU/WeeklyLog Back to Weekly Log] | [https://2012.igem.org/Team:WashU/WeeklyLog Back to Weekly Log] |
Latest revision as of 02:12, 15 September 2012