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+ | <button class="accordion"> <h2> Week 4 (24/7/17-28/7/17)</h2> </button> | ||
+ | <div class="panel"> | ||
+ | <p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <td> <top> <img class="media object pull-left" src="https://static.igem.org/mediawiki/2017/c/c8/Notepadlogo.jpg"> </top> </td> | ||
+ | <td> <h4> Dry Lab </h4> | ||
+ | <ul> | ||
+ | <li> Unofficial team meeting took place as some team members still absent. </li> | ||
+ | <ul> <li> To Do List was discussed. </li> </ul> | ||
+ | <li>There was a meet-up with Ari, Steve, Indi, Louise and Catherine (Pop Up Incubator) where we consult in a game they are designing. </li> | ||
+ | <li> Discussion around potential collaboration opportunities with CSIRO. </li> | ||
+ | <li> Game avatar design finalised and comments shared amongst the team. </li> | ||
+ | <li>Abstract edited for SynBio by India and given to Louise to hand in for registration. </li> | ||
+ | <li> USyd approached to form Australasian Meeting for iGEM-they paid, we dont want to. Need to find out if UMelb and Auckland (other invitees) have paid-if not allows us leverage. </li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | <tr> | ||
+ | <td> <top> <img class="media object pull-left" src="https://static.igem.org/mediawiki/2017/8/81/T--Macquarie_Australia--flasklogo_.png" alt="flasklogo" style= "height:50%; width:50%"> | ||
+ | </td> | ||
+ | <td> <h4> Wet Lab </h4> | ||
+ | <ul> | ||
+ | <li> We were ready to re-attempt the three day plan which starts with 3A assembly ligation of the lac-ferredoxin- FNP with the lac-hydrogenase. </li> | ||
+ | <ul> | ||
+ | <li> As these were both in CAM backbones, they were digested then ligated into Amp backbone. </li> | ||
+ | <li> We used new incubation settings for ligation which ramped up the temperature based on advice from Rob. </li> | ||
+ | <li> Transformed cells were grown on plates overnight. </li> | ||
+ | <ul> | ||
+ | <li> We were excited to achieve one colony of our Fer/Hyd plasmid cells however with very low numbers even on the puC19 positive control plates the competency of the cells was in question.</li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> At the same time we ligated the newly arrived biobricks- GUN4, HydG and Double terminator into CAM backbones. </li> | ||
+ | </ul> | ||
+ | <li> These were also transformed into cells and successfully grown overnight. </li> | ||
+ | <li> Subcultures of these as well as the one Fer/Hyd colony were grown in liquid media ready for miniprep. </li> | ||
+ | </ul> | ||
+ | <li> PCR (both KOD and Q5) was attempted again on all three backbones and the gel showed disappointing results. </li> | ||
+ | <ul> | ||
+ | <li>Having discussed this with Rob it was decided that our templates were a poor choice (iGEM linear backbones). </li> | ||
+ | <li> Our next attempt to amplify the backbones- CAM, Amp and Kan utilised the RFP/antibiotic resistant backbone plasmids from previous iGEM members. </li> | ||
+ | </ul> | ||
+ | <li>While running PCR products on gels we had the opportunity to tweak our NEB 1kb ladder. </li> | ||
+ | <ul> | ||
+ | <li> The ladder used was a 1:10 dilution of the original now with loading dye added to the mix so we could confidently just drop 2uL in a well and see a great result every time. </li> | ||
+ | </ul> | ||
+ | <li> In an attempt to test the competency of the cells we’d been using we challenged Thi’s cells against Rob’s cells (all DH5⍺). </li> | ||
+ | <ul> | ||
+ | <li> We transformed each with our Fer/Hyd ligation and with puC19 +ve control. </li> | ||
+ | <li> As we plated up the cells someone noticed that our heat block temp was not set correctly so effectively our transformants had never been heat shocked. </li> | ||
+ | <ul> | ||
+ | <li> Given these probably won’t work we re-did the transformation. </li> | ||
+ | </ul> | ||
+ | <li> The next day we found those that weren’t heat shocked did grow very few cells for the puC19 +ve control and none for Fer/Hyd. </li> | ||
+ | <li> Rob’s cells showed more colonies. 3 colonies for Fer/Hyd. </li> | ||
+ | </ul> | ||
+ | <li> We had also grow one more colony on a plate from spin down of the previously transformed Fer/Hyd cells which had be held in reserve in the fridge.</li> | ||
+ | <li> Sent off for sequencing were- lacHydG, Double terminator, Gun4 and our Fer/Hyd construct.</li> | ||
+ | <li> That was the last week of what was fondly known as bootcamp. Now that the vacation period is over we will be challenged with university classes and other challenges to our lab time. </li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </p> | ||
</div> | </div> | ||
Revision as of 09:30, 14 October 2017
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