Team:UIUC-Illinois/Notebook/LabNotebook

From 2012.igem.org

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                     <li><a name="week8puf" >Week 8</a></li>
                     <li><a name="week8puf" >Week 8</a></li>
                     <li><a name="week9puf" >Week 9</a></li>
                     <li><a name="week9puf" >Week 9</a></li>
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                    <li><a name="week10puf" >Week 10</a></li>
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                    <li><a name="week11puf" >Week 11</a></li>
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                    <li><a name="week12puf" >Week 12</a></li>
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                    <li><a name="week13puf" >Week 13</a></li>
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                    <li><a name="week14puf" >Week 14</a></li>
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                    <li><a name="week15puf" >Week 15</a></li>
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                    <li><a name="week16puf" >Week 16</a></li>
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                    <li><a name="week17puf" >Week 17</a></li>
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                    <li><a name="week18puf" >Week 18</a></li>
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                    <li><a name="week19puf" >Week 19</a></li>
             </div>
             </div>
                  
                  
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<div id="week9puf" style="display:none">
<div id="week9puf" style="display:none">
<h2>Week 9 notes</h2>
<h2>Week 9 notes</h2>
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<br/>
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<table border=1>
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  <tbody>
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      <td>7/23/12 </td>
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      <td>-Alkanes arrived from Sigma-Aldrich. Isiah plated K12-Petrobrick cells on a CM plate (from glycerol stock) to grow more colonies. Divya made 4 innoculations of K12 cells (one of them accidentally has more than the required cells in it).<br />-Bob did PCR of 3 reactions of cCFP and Uros continued with PCR cleanup once it was done. Uros also amplified the two parts for PUF(WT)-cCFP tethering through PCR. After PCR was done Uros performed PCR cleanup. Made a plan for Bob to take over Split-CFP cloning which includes bypassing gel purification due to low concentrations of vector/insert.<br />-Transformed ligation reactions from 7/19/12 to 65 ul DH5a. Unfortunately electroporation of the 1:3 reaction arced. Transformed using heat shock for both 1:3 and 1:6 ligation reaction. Plated transformations. Started two new reactions using the same volumes as the 7/19 ligations (a 1:3 in 15 ul and a 1:6 in 20 ul)</td>
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    </tr>
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    <tr>
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      <td>7/24/12</td>
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      <td>-The K12 innoculations did not grow because Divya accidentally grew them with CM. Isiah took out the K12-Petrobrick plated cells; they grew successfully. TB media was made, and the filter solution was prepared as well (needs to be autoclaved, however). Divya re-did innoculations of K12, and prepared innoculations of K12-Petrobrick.<br />-Performed PCR in order to tether PUF(WT)-cCFP with GC buffer and the PCR failed, presence of primer dimers. PCR of the amplification of cCFP and PUF for tethering seemed to have worked although there was low cCFP product. The order of PCR for tethering needs to be reversed and optimized tomorrow. Inoculated two tubes of pETDuet-1 for further cloning work.<br />-Desalted the ligations from yesterday and transformed them into DH5a. Plated the transformations. Started two new digestions of pBAD and a digestion of PUF PCR.</td>
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    </tr>
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    <tr>
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      <td>7/25/12</td>
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      <td>-Isiah made minipreps of both K12 and K12-Petrobrick innoculations. We prepared 2 tubes of K12 TB cultures to grow overnight.<br />-Did primer extension in order to tether PUF-cCFP with HF buffer and PCR didn't work, presence of primer dimers. Performed PCR of PUF-cCFP under a gradient of temperatures 50/55/60/66/70oC with an annealing time of 1 minute instead of 30 seconds. PCR was done with WT-PUF, cCFP, and primers PUF-cCFP tether 1F/1R.<br />-Found no colonies on the plates. Also ran a gel of an uncut miniprep of pBAD with one of the pBAD digestions from yesterday. No bands for either.</td>
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    </tr>
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    <tr>
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      <td>7/26/12</td>
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      <td>-11 reactions for wtPuf was made for PCR, 7 was ran today the rest will run tomorrow. Starting over on pSB1C3 tomorrow due to strange results. Divya accidentally put miniprep in TB cultures, so she redid TB culture prep (2 tubes K12; 2 tubes K12-Petrobrick) and set them to grow overnight<br />-Presence of primer dimers under all temperatures of the PCR done yesterday. Ran another PCR for PUF(+)-cCFP tethering with PUF(+) including primers PUF-cCFP tether 1R/PUF-cCFP 1F and cCFP with primers PUF-cCFP tether 1F/PUF-cCFP 1R in order to amplify the parts before tethering. Made 4 inoculations of pETDuet-1 with Bob.</td>
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    </tr>
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    <tr>
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      <td>7/27/12</td>
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      <td>-Found pSB1C3 vectors, ran a gel of pCR and digest of pSB1C3; BOTH WORKED! Took TB cultures out of the 37 degree; EVERYTHING GREW!<br />-Ran a gel of yesterday’s PCR due to taken gel not being clear. The PCR for both PUF(+) and cCFP amplification resulted in primer dimers. Ran PCR cleanup for Bob's digestion of pETDuet-1 and ligated with a 3:1 and No Insert at 15fmole concentrations and left it to incubate overnight.</td>
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    </tr>
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    <tr>
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      <td>7/28/12</td>
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      <td>-Made M9 media, resuspended k12 TB cultures in M9 media and set to grow overnight (1 tube /6 ml) and also injected 75 mg C15 alkanes. Digested wtPUF; Did a gel purification of both wtPUF and pSB1C3. started ligation</td>
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    </tr>
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  </tbody>
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</table>
</div>
</div>
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<div id="week10puf" style="display:none">
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<h2>Week 10 notes</h2>
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<br/>
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<table border=1>
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  <tbody>
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      <td>7/30/12</td>
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      <td>-Colonies grew, but we have to replate transformations because we lost colonies due to Colony PCR we weren't supposed to do. Ran a gel anyways; A BAND WORKED! (L1 - 3). Replated more L1, L2 Transformations to use for colony PCR tomorrow.<br />-Started two new digestions of pBAD miniprep from angela and another digestion of PUF PCR. Used newly bought nuclease free water for these reactions.</td>
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    </tr>
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    <tr>
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      <td>7/31/12</td>
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      <td>-Isiah ran a colony PCR and restreaked colonies on a gridded plate. Divya ran the gel; no bands showed. Talked to Dr. Jin about GCMS; have to regrow cultures in order to have enough samples for a standard curve. Restreaked two colonies from original transformation plates on a gridded plate, innoculated 2 colonies from Asha/Anthony's PUF-YFP/protet colony plates, transformed remaining ligation (L3) and streaked onto a separate plate, replated remaining L1/L2 transformations. Isiah made 5 K12 TB cultures.<br />-Talked to Kori as well as my graduate student about test expression of PUF. Finished test expression plan details. Received Arabinose from Kori.</td>
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    </tr>
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    <tr>
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      <td>8/1/12</td>
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      <td>-The L3 transformation did not grow any colonies; Isiah ran colony PCR on all the remaining plates, but no colonies grew. Fortunately, he streaked colony 3 from the original L1 plate on to a new grid. Divya made 3 innoculations of that colony in 0 CM, CM10, and CM20. The K12 TB cultures were resuspended in M9 media and put for incubation. Divya added CM20 to 5 K12-Petrobrick TB cultures and put them in the 37 degree to grow overnight. Divya made 4 different dilutions of C15 alkanes and injected in the control K12 M9 cultures (1 mg/L, 10 mg/L, 50 mg/L, and 100 mg/L).<br />-Inoculated 6 cultures (3 for WT PUF and 3 for PUF in pBAD). Allow for 16 hours of growth</td>
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    </tr>
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    <tr>
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      <td>8/2/12</td>
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      <td>-Isiah made glycerol stocks and minipreps of the innoculated L1 3 colony and prepared the sequencing bag. Everything was sent for sequencing. Divya took out K12-Petrobrick TB cultures, spun them down and resuspended in M9 media to grow for 48 hours.<br />-Sucultured 50ul of each PUF culture into 5mL of LB. Grow for 2 hrs for PUF in pBAD, and 3 hrs for WT PUF. Induced with Arabinose and IPTG for corresponding cultures. Grow for 16 hours</td>
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    </tr>
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    <tr>
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      <td>8/3/12</td>
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      <td>-Divya took out K12-M9 cultures which had been injected with alkanes and centrifuged them. Ethyl Acetate was added to extract 200 uL of the top layer after centrifuging it. The samples were collected in 4 1.5 mL Eppendorf tubes. Dr. Jin said to talk to Na Wei on Monday to make time to run the samples through GCMS<br />-Pellet cells, resuspend in 700ul Binding buffer. Lyse cells by sonication. Run gel of pellets and lysates (12 samples). Stain and destain gel. Protein gel does not show clear expression of PUF in any of the samples.</td>
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    </tr>
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    <tr>
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      <td>8/4/12</td>
 +
      <td>-Divya came back at night to take out the K12-Petrobrick M9 cultures. She centrifuged the samples after adding .7 mL of Ethyl Acetate and noticed that one sample had a particularly high amount of cell debris when compared to the other. 2 extractions of 200 uL each was made of the tubes, respectively. Pictures were taken on her phone. These samples, along with the control samples, will be run on the GCMS on Monday<br />-Cotransformed minipreps of PUF in pBAD and YFP-control/YFP-PBS into DH5a cells using 3 different ratios of PUF:YFP. Plate cells on AMP+CM plates. Also transformed PUF in pBAD into YFP-control/YFP-PBS overnight cultures (wash cells in glycerol first). Plate cells on AMP+CM plates.</td>
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    </tr>
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    <tr>
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      <td>8/5/12</td>
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      <td>-Found colonies on Yesterday's plates (cotransformation only). Started digestions for cloning YFP-control and YFP-PBS into pET-duet. Subcultured yesterdays PUF overnights (1/100 dilutions again). Grew PUF in pBAD for 2 hrs before inducing Arabinose. Grew WT PUF for 3 hrs before inducing with IPTG. Allow to express for 16 hrs (in 20 degrees this time).</td>
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    </tr>
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  </tbody>
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</table>
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</div>
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<div id="week11puf" style="display:none">
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<h2>Week 11 notes</h2>
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<table border=1>
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      <td>8/6/12</td>
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      <td>-Pellet cells, resuspend in 700ul Binding buffer. Lyse cells by sonication. Run gel of pellets and lysates (12 samples). Stain and destain gel. Protein gel does not show clear expression of PUF in any of the samples. Growing in 20 degrees seemed to reduce expression of all proteins.</td>
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    </tr>
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    <tr>
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      <td>8/7/12</td>
 +
      <td>-Made a plan for further RNA scaffold work.<br />-Run colony PCR on Asha's cotransformations. Only one colony (Had YFP-contol band) showed a band. Tested amp and CM stocks on DH5a to ensure quality. All tested ones worked.</td>
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    </tr>
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    <tr>
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      <td>8/8/12</td>
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      <td>-Designed and ordered primers for the RNA scaffold. An addition of EcoRI will be made at the 5' site in order to clone into a T7 promoter vector (pETDuet-1). Sent a draft of PUF(+)-(a)GFP for synthesis to Brad in order to receive a final review before sending out.<br />-Made m9 minimal media (with glucose) as well as m9 media plates. Autoclaved 6 boxes of p1000 tips, 6 boxes of p10 tips, 4 boxes of p200 tips. Made 500 mL of LB media. Inoculated 4 cultures of PUF in BL21 for test expression.</td>
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    </tr>
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    <tr>
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      <td>8/9/12</td>
 +
      <td>-Received an improved version of the PUF(+)-(a)GFP draft from Brad, the 6x His Tag was changed. Sent the updated and final sequence to Courtney in order to get a quote and purchase.<br />-Made Amp+Cm plates. Made new stock of Cm 34mg/ml and Amp 100 mg/ml. Missed inducing time, will need to start inoculations again. Inoculated 6 cultures of PUF WT in BL21.</td>
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    </tr>
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    <tr>
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      <td>8/10/12</td>
 +
      <td>-Performed quality control on M9 liquid media (grow Dh5a), M9 plate (grew PUF in pBAD, DH5a, YFP-control, colony 3 ), Cm20 plate (grew DH5a)<br />-Subcultured cells (1/100) in 5 mL, induced at OD600 of 0.6-0.8 with 6 different IPTG concentrations. Performed quality control on M9 liquid media (grow Dh5a), M9 plate (grew PUF in pBAD, DH5a, YFP-control, colony 3 ), Cm20 plate (grew DH5a)</td>
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    </tr>
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    <tr>
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      <td>8/11/12</td>
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      <td>-re-inoculate biobrick. Get grid plate and innoculate. Remake M9 media using Ahmet protocol. Miniprep and test colony 3 by running gel. Transform mutant PUF. Inoculate pKRSTc or streak a plate. Make tet12 and Kan40 plate. Buy DH5a comp cells. Miniprept WT PUF and PUF biobrick fo sequencing. Prepare strong RBS SOE. Make more YFP (control/PBS). Kitplate 1 well 16P, tetR repression transformation. Plate 2 (3K)<br />-Pellet, resuspend cells, sonicate, run gel, stain and destain gel. There seems to be a band the corresponds to the molecular weight of PUF but it is still relatively feint and is present in every lane.</td>
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    </tr>
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    <tr>
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      <td>8/12/12</td>
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      <td>-Co transformed minipreps of: 1) protet (empty) + PUF in pBAD 2) YFP-control + PUF in pBAD 3) YFP-PBS + PUF in pBAD. Inoculated colony 3, PUF biobrick, and PUF in pBAD</td>
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    </tr>
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  </tbody>
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</table>
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</div>
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<div id="week12puf" style="display:none">
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<h2>Week 12 notes</h2>
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<table border=1>
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      <td>8/13/12</td>
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      <td>-Prepared 20 plates of Kanamycinand 20 plates of Kanamycin + Tetracycline<br />-Found colonies in all plates, did colony PCR of colonies. Inoculated E0030 and PUF biobrick. Transformed mutant PUF into BL21 DE3, plate on Kan plate.</td>
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    </tr>
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    <tr>
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      <td>8/14/12</td>
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      <td>-Made the final draft of PUF(6-2/7-2)-BGFP and sent to Brad in order to confirm before synthesis. Made a wetlab plan that spans till the regional competition.<br />-Ran gel of colony PCR, found 2 colonies that worked. Transformed TetR repressor into DH5a, plate on kan plate. Inoculate tubes for YFP fluorescence test: DH5a, Protet, YFP-control, YFP-PBS, Protet+PUF(pBAD), YFP-control+PUF (pBAD), YFP-PBS+PUF (pBAD).</td>
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    </tr>
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    <tr>
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      <td>8/15/12</td>
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      <td>-Prepared IDTE buffer for resuspension of the RNA scaffold.<br />-Subcultured YFP test cultures into 2 sets (37 and 24 degrees expression) as well as separate induced/uninduced for the appropriate tubes (20 tubes total, 10 tubes per set). Induced the appropriate tubes after 2 hrs, and moved tubes into their respective expression temperatures. Measured fluorescence of each culture after 6 hrs using plate reader. Inoculated WT PUF and mutant PUF for first test expression set. Inoculated pBAD PUF, YFP-control+PUF (pBAD), YFP-PBS+PUF (pBAD) for second set of test expression.</td>
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    </tr>
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    <tr>
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      <td>8/16/12</td>
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      <td>-Sent out PUF(6-2/7-2)-(B)GFP to get a quote from GenScript. Studied the in-vitro transcription protocol and followed a graduate student perform the procedure.<br />-Ran day 2 test expression of mutant PUF. Subcultured and induced at various IPTG concentrations. Grow cultures overnight, one set at 24 degrees and another at 37 degrees. Ran day 2 test expression for YFP constructs as well. subcutured and induced the appropriate tubes with 50ul 20% arabinose. Grew at room temperature for 6 hrs. Pelleted, resuspended, sonicated, ran gel, stain/destain gel.</td>
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    </tr>
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    <tr>
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      <td>8/17/12</td>
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      <td>-Sent Courtney the quote received from GenScript<br />-Ran day 3 test expression for mutant PUF. Pellet, resuspend cells, sonicate, run gel, stain and destain gel. There is a clear indication that mutant PUF is expressed</td>
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    </tr>
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  </tbody>
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</table>
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</div>
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<div id="week13puf" style="display:none">
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<h2>Week 13 notes</h2>
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      <td>8/20/12</td>
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      <td>-Diluted the RNA scaffold in IDTE buffer to make a final concentration of 50ng/uL. Transformed the scaffold into DH5a cells and plated on an ampicillin plate.</td>
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    </tr>
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    <tr>
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      <td>8/22/12</td>
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      <td>-Inoculated four tubes of 5mL LB with DH5a RNA Scaffold cells. Miniprepped the four tubes and received low concentrations so inoculated a 500mL flask with 20mL of LB.</td>
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    </tr>
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    <tr>
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      <td>8/23/12</td>
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      <td>-Miniprepped 10mL of the flask inoculated last night and received very low concentrations. Miniprepped the remainder of the inoculation using N3 buffer instead of P3 buffer and received 650ng/uL.</td>
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    </tr>
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    <tr>
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      <td>8/24/12</td>
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      <td>-Digested 6.5ug of the miniprepped RNA scaffold with PstI for 7 hours. Ran a gel of the digest to gel purify and received a large smear in a range of unexpected lengths. Ran a gel to confirm plasmid integrity and showed expected structure therefore digestion will be ran for less due to star activity. Inoculated 50mL of LB with DH5a RNA scaffold transformed cells.</td>
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    </tr>
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    <tr>
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      <td>8/25/12</td>
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      <td>-Miniprepped 10mL of the 50mL inoculated flask and received 72ng/uL. Miniprepped in RAL and received a similar concentration of 82ng/uL. The concentrations are too low to get 6ug of DNA so made a 20mL inoculation of the RNA scaffold cells for tomorrow.</td>
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    </tr>
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  </tbody>
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</table>
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</div>
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<div id="week14puf" style="display:none">
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<h2>Week 14 notes</h2>
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      <td>8/27/12</td>
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      <td>-Miniprepped 10mL of the remainder of the 20mL inoculation and received 210ng/uL. Digested 4.8ug of DNA with PstI-HF for 4 hours with addition of CIP after 1 hour of incubation. <br />-Proceeded to gel purify the band which was at the expected length and received 30ng/uL. The concentration was low, but started in-vitro transcription with an overnight incubation to ensure product formation.</td>
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    </tr>
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    <tr>
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      <td>8/29/12</td>
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      <td>-Eluted RNA from the in-vitro transcription ran overnight following a more extensive and detailed protocol. Quantified the RNA and received decent concentrations of approximately 30ng/uL in a 30uL stock. <br />-The A260/280 values were abnormal, however, since there is presence of RNA, a denaturing gel should be done in order to check the length in order to assess desired purity. <br />-Inoculated a 250mL flask with the RNA scaffold in 50mL of LB if more DNA template is needed to further isolate RNA.<br />-Reinoculated the same cultures as yesterday since subculure time was missed. Transformed LacZ-WTPUF and LacZ-mutant PUF into DH5a and plated on amp plates.</td>
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    </tr>
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    <tr>
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      <td>8/30/12</td>
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      <td>-To further test the RNA purified from the previous two in-vitro transcriptions a 10% denaturing acrylamide gel was cast. The two products were ran with RNAase free loading dye and NEB ssRNA Low Range Ladder. <br />-The two products were pure, one containing a higher concentration of RNA, although greater presence of degraded parts. The ladder shows the products are approximately 150bp, however, the ladder was not heated before loading into the gel therefore presence of secondary structure would account for the lower length seen.<br />-Subcultured yesterday's cultures (1/100). Induced with arabinose after 2 hrs. Read fluorescence after 4 hrs. Inoculate the same tubes as yesterday for another trial. Inoculated one tube of BL21 DE3, LacZ+WTPUF from transformation plate, and LacZ+MutantPUF from transformation plate.</td>
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    </tr>
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    <tr>
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      <td>8/31/12</td>
 +
      <td>-Constructed a plan for further scaffold work which mainly consists of purifying PUF(+/-) proteins in order to run further gel-shift assays.<br />-Subcultured yesterday's cultures (1/100). Induced with arabinose after 2 hrs. Read fluorescence after 4 hrs. Make glycerol stocks of Asha's cotransformation of mutantPUF+YFP-PBS/control. Made new BL21 DE3 and DH5a competent cells</td>
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    </tr>
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  </tbody>
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</table>
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</div>
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<div id="week15puf" style="display:none">
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<h2>Week 15 notes</h2>
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<table border=1>
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      <td>9/6/12</td>
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      <td>-Reviewed papers for insight into how PUF was purified. Looked through past protocols and planed a rough outline for purification.</td>
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    </tr>
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    <tr>
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      <td>9/7/12</td>
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      <td>-Consulted Brad about purification and need for supplies. Researched further into PUF purification. Confirmed with Dr. Jin that he might have proper purification supplies.</td>
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    </tr>
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    <tr>
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      <td>9/8/12</td>
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      <td>-Performed PCR cleanup and quantified tubes for Angela.</td>
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    </tr>
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    <tr>
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      <td>9/9/12</td>
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      <td>-Transformed and inoculated for Angela. Plated WT PUF-PIN on an Amp plate and mutant PUF-PIN on a Kan plate after Angela transformed the plasmids (pET4.3) into BL21 cells.</td>
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    </tr>
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  </tbody>
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</table>
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</div>
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<div id="week16puf" style="display:none">
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<h2>Week 16 notes</h2>
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<table border=1>
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      <td>9/10/12</td>
 +
      <td>-Inoculated 25mL of WT PUF-PIN and 6-2/7-2 PUF-PIN for protein purification experiments. Concurrently, prepared two separate 1L LB medias for purification of each protein and another 1L of LB for general iGEM use. Inoculated 3mL of WT PUF-PIN in pBAD for Divya.<br />9/12/12:<br />-Inoculated two 1L bottles of LB media with 5mL of WT PUF-PIN overnight in one and 5mL of 6-2/7-2 PUF-PIN overnight in the other after adding 1mL of Ampicillin to each media at 3:30PM. Incubated at 37oC and checked OD levels at 4:00PM when there was no change in OD of the cultures from standard LB media. <br />-Checked the OD levels again at 5:12PM and received 0.0419 for the WT PUF-PIN. It was at this time that it was observed that the 6-2/7-2 culture wasn't growing like the WT PUF-PIN culture was. This was due to Ampicillin being added to the culture rather than Kanamycin. <br />-Checked OD levels at 5:38PM and received 0.0902. Checked again at 7:07PM and received a value of 0.5558, at which time 2mM of IPTG was added. After 2 hours of incubation, the cells were spun down at 10000g for 15 minutes. The pellet was then resuspended and centrifuged multiple times in one 50mL conical tube at 6000g for 5 minutes until there was a single pellet.</td>
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    </tr>
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    <tr>
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      <td>9/12/12</td>
 +
      <td>-Inoculated 1L of LB media with 5mL of 6-2/7-2 PUF-PIN overnight after adding 1mL of Kanamycin at 12:15PM. After incubating at 37oC, checked OD levels at 3:00 and received a measure of 0.0275. Checked again at 5:25PM and received 0.3509. <br />-Checked once more at 6:07PM and received a value of 0.5427 and proceeded to add 2mM of IPTG. Incubated the media for 2 hours and then spun down cells at 10000g for 15min. <br />-Resuspended the cells and kept transferring culture into one 50mL conical tube until one pellet was formed. Autoclaved water and other glassware in order to prepare buffers for protein purification.</td>
 +
    </tr>
 +
    <tr>
 +
      <td>9/13/12</td>
 +
      <td>-Performed PCR cleanup and quantified tubes for Angela.</td>
 +
    </tr>
 +
    <tr>
 +
      <td>9/14/12</td>
 +
      <td>-Prepared buffers for protein purification which includes 500 mL of 25mM Tris-Cl at pH8, 500 mL of 0.5 M NaCl, and 50 mL of 0.1% Triton-X. Autoclaved numerous 100 mL bottles in order to serve as containers for a gradient of wash buffers and a final elution buffer (25, 70, 115, 160, 205, and 250 mM concentrations). <br />-Transformed WT PUF-aGFP into both DH5a and BL21 and plated on Ampicillin plates which were left to incubate.</td>
 +
    </tr>
 +
    <tr>
 +
      <td>9/16/12</td>
 +
      <td>-Inoculated 25 mLs of LB media with WT PUF-aGFP in BL21 and left to incubate overnight.</td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
</div>
 +
 +
 +
<div id="week17puf" style="display:none">
 +
<h2>Week 17 notes</h2>
 +
<br/>
 +
<table border=1>
 +
  <tbody>
 +
    <tr>
 +
      <td>9/17/12</td>
 +
      <td>-Prepared the final wash and elution buffers for protein purification with a gradient of concentrations 20, 40, 60, 80, 100, and 500 mM imidazole. Prepared the lysis buffer and stored all buffers in the 4oC room. <br />-Due to absence of literature for purification of PUF-a/BGFP, three vials of 200 mL LB media were inoculated with 2 mL of overnight and set for incubation at different induction times (2 hr., 6hr., and overnight). <br />-Inoculated at 1:00PM and checked the OD levels at 2:40PM and received a value of 0.0916 nm. Checked again at 4:43PM and received a value of 1.0831 nm. At 4:55PM the vials were induced with 2 mM IPTG. Once the 2 hr. and 6 hr. induction was done it was spun down into a pellet and stored in the freezer.</td>
 +
    </tr>
 +
    <tr>
 +
      <td>9/18/12</td>
 +
      <td>-Spun down the WT PUF-aGFP inoculation which was induced for 31.5hrs. into a pellet by 4 cycles of 4000g for 5 minutes at 4oC.</td>
 +
    </tr>
 +
    <tr>
 +
      <td>9/19/12</td>
 +
      <td>-Resuspended all pellets by using 13 mLs of lysis buffer and sonicated for 6 intervals of 30 sec. at setting 3. Then spun down the suspension at 35000g for 30min. at 4oC. Meanwhile, cast a 6% acrylamide gel which will be used for SDS-PAGE to assess production of protein in each pellet. <br />-Heated 5 uL of lysate sample with 5 uL of SDS in 60oC water for 15 minutes and then ran the samples with 2 uL of BioRad Kaleidoscope Protein Plus Ladder diluted in 8 uL of H20 for 40 minutes at 240V. Stained the gel for 20 minutes with a coomassie stain and destained with H20 for 1 hour.</td>
 +
    </tr>
 +
    <tr>
 +
      <td>9/21/12</td>
 +
      <td>-Thawed frozen lysates of WT PUF-PIN and 6-2/7-2 PUF-PIN. Proceeded to cast a 6% 15 well acrylamide gel to test for protein purity after purification. Added 1 mL of Ni-NTA to the WT PUF-PIN lysate and attached it to the vortex to spin for 1 hour in the 4oC room. Then the protein purification stand was set up and the lysate poured into the purification column. A cap was placed over the column and a syringe used to push the lysate through. After the lysate passed, 20 mL of wash buffers of gradients 20, 40, 60, 80, and 100 mM imidazole were used to wash away unspecified proteins all the while using the syringe and collecting the fractions in 1.5 mL centrifuge tubes. Finally, 20 mL of 500 mM imidazole were used to wash the his-tagged protein and the entire volume was collected in centrifuge tubes. Then 5 uL of 1x SDS loading dye were added to 5 uL of each fraction and let to incubate in 60oC water for 20 min. <br />-Meanwhile, the cast gel was pre-run for 20 min. on 240V before samples were added. Once the gel was prepped, the samples were added and ran for 40 min. at 240V. The gel was then stained with coomassie stain for 1 hr. and destained with H20 for 1 hr. After the gel was finished, it was imaged on GelDoc XR along with the SDS-PAGE gel of lysates from 9/19. While the gel was running, 1 mL of used Ni-NTA were added to 6-2/7-2 PUF-PIN and left to shake in the 4oC room. The same procedure for purification was followed and another SDS-PAGE gel was ran to test for purity of the protein collected.</td>
 +
    </tr>
 +
    <tr>
 +
      <td>9/23/12</td>
 +
      <td>-Inoculated 1 L of LB media with 5 mL of WT PUF-aGFP with 1 mL of Ampicillin and incubated at 37oC at 2:30PM to prep for protein purification. <br />-Checked the OD levels at 6:20PM and received a reading of 0.5530nm. Decided to incubate for longer and checked the OD at 6:50PM where it was at 0.6814nm and induction with 2 mM IPTG was done at 6:55PM. The culture was left to incubate in the 37oC room overnight.</td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
</div>
 +
 +
<div id="week18puf" style="display:none">
 +
<h2>Week 18 notes</h2>
 +
<br/>
 +
<table border=1>
 +
  <tbody>
 +
    <tr>
 +
      <td>9/24/12</td>
 +
      <td>-After 22 hours of induction, the culture was spun down in 500 mL flasks at 10000g, 4oC, for 15 min. The pellets were then resuspended and spun down again in 50 mL conical tubes at 4000g, 4oC for 5 minutes until one pellet was formed.</td>
 +
    </tr>
 +
    <tr>
 +
      <td>9/25/12</td>
 +
      <td>-A 10% denaturing acrylamide gel was cast in preparation for the endonuclease assay. Due to literature referencing PIN working best with Mn2+ ions, different ion concentrations were prepared (3mM concentrations of MnO2, MnCl2*4H20, and MgCl2 were made). While the gel was casting, the reactions were prepared. <br />-There were 12 reactions, with three sets of 4 different conditions (RNA control, RNA+WT PUF-PIN, RNA+6-2/7-2 PUF-PIN, and RNA+WT PUF-PIN+6-2/7-2 PUF-PIN) with the three sets comprised of the three different ions used. The amounts of each reagent include 5 uL of each protein (or H20 when not included), 2 uL of RNA, 2 uL of loading dye, and 1 uL of ions ~ 15 uL reaction. The reactions were incubated in 37oC for 30 minutes and then loaded into the gel once the gel was pre-ran. The gel was ran for 40 min. at 240V with 1X TBE buffer. <br />-Once the gel was done, it was stained in 10 ug/mL EtBr for 20 min. and destained in H20 for 20 min. whereupon it was imaged with GelDoc XR. The gel image showed no signs of RNA so new a new 10 ug/mL EtBr stain was prepared and the gel was stained again for 20 min. followed by a 20 min. destain with H20. The gel was imaged again and there was were no signs of RNA.</td>
 +
    </tr>
 +
    <tr>
 +
      <td>9/27/12</td>
 +
      <td>-Due to the endonuclease assay failing, the RNA and protein preparations were tested. A 15 well 6% SDS-PAGE was ran for testing WT PUF-PIN and 6-2/7-2 PUF-PIN. In order to test the RNA viability a 10% urea denaturing gel was prepared.</td>
 +
    </tr>
 +
    <tr>
 +
      <td>9/28/12</td>
 +
      <td>-The protein SDS-PAGE gel was stained with coomassie blue for 45 min. and destained overnight.</td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
</div>
 +
 +
<div id="week19puf" style="display:none">
 +
<h2>Week 19 notes</h2>
 +
<br/>
 +
<table border=1>
 +
  <tbody>
 +
    <tr>
 +
      <td>10/1/12</td>
 +
      <td>-A 10% Denaturing 10 well acrylamide gel was cast. A 30mM MnCl2*4H20 pH 8.25 buffer was made. 1 uL of RNA was added to 5 uL of RNAse free H20 and 1 uL of annealing buffer (**EDTA, Tris). The RNA was then denatured at 95oC for 3 min. and let to fold for 5 min. at 4oC. <br />-Then 2 uL of protein was added (WT, 6-2/7-2 PUF-PIN) to the respective tubes followed with 1 uL of either 30 mM MnCl2*4H20 or 30 mM MgCl2. The tubes were then incubated at 37oC for 30 min. Afterward, 10 uL of 80% formamide, EDTA/Tris were added to the tubes. The tubes were let to sit for another 30 min. while the gel was pre-run at 120V for 25 min. <br />-The samples were then loaded and the gel was let to run at 120V for 55 min. The gel was then stained in 2 ug/mL EtBr for 20 min. and destained in H20 for 20 min.</td>
 +
    </tr>
 +
    <tr>
 +
      <td>10/3/12</td>
 +
      <td>-A BCA analysis was done in order to determine the concentrations of both WT & 6-2/7-2 PUF-PIN. A 10% Denaturing 10 well acrylamide gel was cast. 1 uL of annealing buffer was added to each tube for the endonuclease assay followed by 1 uL of 66 uM RNA. <br />-Then the proper amounts of RNAase free H20 were added to each tube with the overall volume of the reactions being 10 uL. The RNA was then denatured at 95oC for 3 min. and let to refold in the 4oC for 5 min. Then 300 nM concentrations of proteins were added to the first 4 tubes while 150 nM concentrations of protein were added to the last 4. 1 uL of 30 mM MgCl2 was added to each reaction and they were let to incubate at 37oC for 30 min. <br />-Then 2X 80% formamide/EDTA was added to each tube and loaded onto the pre-ran gel at 120V for 55 min. The gel was then stained with 2 ug/mL EtBr for 30 min. and destained for 20 min. followed with imaging.</td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
</div>
 +
</div>
</div>

Latest revision as of 05:14, 26 October 2012

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