Team:UIUC-Illinois/Notebook/LabNotebook

From 2012.igem.org

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       <td>7/16/2012</td>
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       <td>7/16/12 </td>
       <td>- Ordered 16 primers for construction of pETDuet with split-CFP parts and the tethering of PUF-CFP (primer extension). <br />- 4 primers were constructed by Angela for the sequencing of MSC1 and MSC2 of pETDuet-1.<br />- Inoculated two cultures from the transformation plate (one each from the 7/7 ligation and 7/10 ligation. The culture did not grow. <br />- Reinoculated four cultures (same as before except duplicates of each with no ampicillin). <br />- Also streaked competent cells on an amp plate. Did this to see if it is problem regarding ampicillin concentration. <br />- Also started two new digestions of PUF and pBad for and construct.</td>
       <td>- Ordered 16 primers for construction of pETDuet with split-CFP parts and the tethering of PUF-CFP (primer extension). <br />- 4 primers were constructed by Angela for the sequencing of MSC1 and MSC2 of pETDuet-1.<br />- Inoculated two cultures from the transformation plate (one each from the 7/7 ligation and 7/10 ligation. The culture did not grow. <br />- Reinoculated four cultures (same as before except duplicates of each with no ampicillin). <br />- Also streaked competent cells on an amp plate. Did this to see if it is problem regarding ampicillin concentration. <br />- Also started two new digestions of PUF and pBad for and construct.</td>
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       <td>7/17/2012</td>
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       <td>7/17/12</td>
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       <td>- Ran a PCR of wtPUF and a gel to go along with it.<br />- Corrected a primer for PUF-cCFP tethering and re-submitted the order. <br />- Inoculated pETDuet-1 and K157005 as well as transformed K157006 into DH5a cells. <br />- Plated the transformation on an ampicillin plate afterword. </td>
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       <td>-Today, we ran a PCR of wtPUF and a gel to go along with it. We will contact Sigma-Aldrich to see when the C15 alkanes are arriving and plan for it. We should also transform pSB1C3 into dH5a.<br />-Corrected a primer for PUF-cCFP tethering and re-submitted the order. Made a detailed plan of the cloning that needs to be done for PUF-CFP tethering and split-CFP construction. Bob inoculated pETDuet-1 and K157005 for me as well as transformed K157006 into DH5a cells. I plated the transformation on an ampicillin plate afterword.<br />-Made new primer sequences for PUF project. Modify YFP construct using Venus gene (proven it worked) instead of biobrick: E0030. A set of CPEC primers are also designed. Transformed the 6:1 ligation of YFP. Also inoculated more protet. For YFP florescence testing will need a negative and a positive control....<br />-Ran gel of digested pBAD and PUF for construct. Only found band for PUF. Second time in a row where digested pBAD showed no bands.Gel extracted the PUF band. Also started three new digestions of pBAD. Checked inoculations, the two without ampicillin grew, but the other two with ampicillin did not grow. Also checked streaked plate of competent cells on amp plate. Found a lawn. Concluded that the amp plates do not have enough ampicillin. Therefore the transformation plates were not reliable.</td>
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      <td>7/18/12</td>
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      <td>-Isiah did a gel extraction as well as ran a gel of Divya's PCRed wtPUF. The PCR was successful as the bands were around 1.6 kb, close to 1.7 kb.<br />-Received the split-GFP sequences used by Mrs. Valencia-Burton in her paper for characterizing an RNA scaffold! The final PUF-GFP sequence or synthesis can be subsequently made to have as backup if tethering doesn't work.<br />-Did PCR cleanup on the three pBAD digests (no gel extraction in order to increase yield). Concentrations were still extremely low. Used pBAD digest with 7/10 and 7/16 PUF digests for two ligation reactions. Also started a digestion of PUF construct PCR and two digestions of pBAD. Increased the DNA mass for the digestions so that concentrations could be high enough after PCR cleanup for different ligation ratios.</td>
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      <td>7/19/12</td>
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      <td>-Isiah did a PCR of wtPUF according to a different protocol. He PCRed five samples. Divya ran a gel of those samples in the afternoon; they were unsuccessful. We shall repeat the pcr tomorrow.<br />-Did PCR of cCFP in order to amplify the band before digestion. PCR proved to work after Bob ran a gel of it. Made a final draft of PUF(WT)-aGFP ready for synthesis in case tethering doesn't work. Made two inoculations of K157006 in preparation for tethering and cloning tomorrow.<br />-Angela miniprepped three plasmids: pBAD30, P. protet. E, and the plasmid encoding venus. A glycerol stock of pBAD30 was made. Ligation of several testing constructs (6 YFP test constructs and 1 PUF binding site and YFP test construct) were completed and sit overnight.<br />-Transformed and plated the ligation reactions from yesterday. Plated two plates for each ligation reaction (100 ul and 200 ul) for a total of four plates. Also did PCR cleanup for the three digestions from yesterday. Yields were a bit better due to greatly increased DNA mass used, but loss of total DNA mass after cleanup/extraction was still severe. Started two ligations with this set of digestions using a 1:3, 15 ul reaction and a 1:6, 20 ul reaction.</td>
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      <td>7/20/12</td>
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      <td>-Ran the gel for wtPUF PCR for PCR clean up. The gel's bands for wtPUF and ladder were squiggly, although it does appear that the bands matched up. As a precaution another wtPUF pcr will be ran. pSB1C3 as well.<br />-Adi miniprepped and nanodropped two tubes of K157006 as well as a tube of K157005 and pETDuet-1 for me. Bob and Adi then ran digestion of cCFP and pETDuet-1 at 1500ng with EcoRI/PstI in NEB Buffer 3 with BSA for 4 hours. I ran a gel of the digestions and extracted the proper bands. Ran gel purification and then let the ligation run overnight with 5:1, 3:1, and No Insert reactions stored in the 4oC.<br />-Checked plates from yesterday, non of the plates had colonies.</td>
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Revision as of 04:36, 26 October 2012

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