Difference between revisions of "Team:Cardiff Wales/Notebook/Text"

Line 93: Line 93:
 
<li>Discuss with water institute about helping other teams for collaborations. </li>
 
<li>Discuss with water institute about helping other teams for collaborations. </li>
 
<li>Email Bayer for human outreach aspect.</li>
 
<li>Email Bayer for human outreach aspect.</li>
−
<li>Set up Lvl 0 reactions for GUS and 8 (digestion/ligation/transformation).</li>
+
<li>Set up Lvl 0 reactions for GUS, ATH, and enhancer (digestion/ligation/transformation). (RC and AT)</li>
 
<li>Transform enhancer from biobrick plate 6 L14.</li>
 
<li>Transform enhancer from biobrick plate 6 L14.</li>
 
<li>PCR all of the miniprepped Lvl 0 constructs to check that they are right- run on a gel. </li>
 
<li>PCR all of the miniprepped Lvl 0 constructs to check that they are right- run on a gel. </li>
Line 111: Line 111:
 
<li>Run PCR products on a gel - there were no bands.</li>
 
<li>Run PCR products on a gel - there were no bands.</li>
 
<li>Discussion about logo design.</li>
 
<li>Discussion about logo design.</li>
 +
<li>GUS, ATH and Enhancer didn't work, so re-performed Dig/Lig (RC and AT).</li>
 
</ul>
 
</ul>
 
<br><br><br><br><br>
 
<br><br><br><br><br>
Line 122: Line 123:
 
<li>Meeting with Dan to discuss 3D printing for human outreach. </li>
 
<li>Meeting with Dan to discuss 3D printing for human outreach. </li>
 
<li>Transformations using the iGEM registry parts for the interlab study. </li>
 
<li>Transformations using the iGEM registry parts for the interlab study. </li>
 +
<li>GUS,ATH, Enh cells grew but were all white. Performed colony PCR (cPCR) anyway but none worked. (RC and AT).</li>
 
</ul>
 
</ul>
 
<br><br><br><br><br>
 
<br><br><br><br><br>
Line 132: Line 134:
 
<li>Colonies with correct band sizes grown overnight.</li>
 
<li>Colonies with correct band sizes grown overnight.</li>
 
<li>Aphids obtained and placed onto tobacco plant.</li>
 
<li>Aphids obtained and placed onto tobacco plant.</li>
 +
<li> Dig/Lig on GUS, ATH and Enh again. Transformed and grew overnight (RC and AT). </li>
 +
<li>Made new plates with surface IPTG (40 microlitres of 100mM) and X-Gal (96microlitres of 25mg/ml) (RC and AT)</li>
 +
<li>Transformed InterLab study constructs (RC, AT and EM) </li>
 
</ul>
 
</ul>
 
<br><br><br><br><br>
 
<br><br><br><br><br>
Line 143: Line 148:
 
<li>Colony PCR for level 1 plates that didn’t show bands in yesterday’s colony PCR.</li>
 
<li>Colony PCR for level 1 plates that didn’t show bands in yesterday’s colony PCR.</li>
 
<li>More seeds of Nicotiana benthamiana were planted and the condition of existing plants was monitored.</li>
 
<li>More seeds of Nicotiana benthamiana were planted and the condition of existing plants was monitored.</li>
 +
<li>ATH and GUS were pale blue so left in fridge over weekend (RC and AT). </li>
 +
<li>Dig/Lig again on GUS, ATH and Enhancer. Transformed these and redid 2N and 2F interlab plates due to poor growth, allowed to grow over weekend (RC and AT).</li>
 +
<li>Picked 2 colonies from each InterLab plate and grew over weekend (RC, AT and EM). </li>
 
</ul>
 
</ul>
 
<br><br><br><br><br>
 
<br><br><br><br><br>
Line 157: Line 165:
 
<li>Contacted iGEM HQ about collaborations.</li>
 
<li>Contacted iGEM HQ about collaborations.</li>
 
<li>Tutorial on how to use plate reader for interlab study</li>
 
<li>Tutorial on how to use plate reader for interlab study</li>
 +
<li>cPCR on InterLab plates (RC, AT, AND EM) </li>
 +
 +
 
</ul>
 
</ul>
 
<br><br><br><br><br>
 
<br><br><br><br><br>
Line 171: Line 182:
 
<li>Transform G7 and 35S terminators from the iGEM registry (plate 6 14N + 16J) and then plate out and grow overnight.</li>
 
<li>Transform G7 and 35S terminators from the iGEM registry (plate 6 14N + 16J) and then plate out and grow overnight.</li>
 
<li>1st Calibration completed for Interlab study, performed at 37°C.</li>
 
<li>1st Calibration completed for Interlab study, performed at 37°C.</li>
 +
<li>Enhancer level 0 worked but GUS and ATH did not. Picked and grew enhancer overnight with good InterLab cells (RC and AT). </li>
 +
<li>Set up level 0 dig/lig with GUS and ATH (RC and AT) </li>
 +
 
</ul>
 
</ul>
 
<br><br><br><br><br>
 
<br><br><br><br><br>
Line 187: Line 201:
 
<li>Grow up 14N and 16J colonies overnight. </li>
 
<li>Grow up 14N and 16J colonies overnight. </li>
 
<li>Grow up GFP and 35S long from the glycerol stocks overnight.</li>
 
<li>Grow up GFP and 35S long from the glycerol stocks overnight.</li>
−
<li>2nd and 3rd Calibration completed, performed at 37°C </li>
+
<li>2nd and 3rd Calibration completed, performed at 37°C. </li>
 +
<li> Entered ILS data into excel (RC). </li>
 +
<li> Performed culture part of ILS, took and measured t0 samples at 10:55, then t6 at 4:55. Measured Abs and Fluorescence (RC and EM). </li>
 +
<li> Miniprepped enhancer (HE).</li>
 
</ul>
 
</ul>
 
<br><br><br><br><br>
 
<br><br><br><br><br>
Line 200: Line 217:
 
<li>Level 1 digest and overnight ligation using RTBVp/Enhancer/GFP/Nos.</li>
 
<li>Level 1 digest and overnight ligation using RTBVp/Enhancer/GFP/Nos.</li>
 
<li>Cell measurement protocol continued, dilution and measurements taken.</li>
 
<li>Cell measurement protocol continued, dilution and measurements taken.</li>
−
<li>Colonies incubated overnight for Interlab study.</li>
+
<li>Colonies incubated overnight for Interlab study CFU section (RC and EM).</li>
 +
<li> Entered ILS data and submitted measurement part (RC) </li>
 
</ul>
 
</ul>
 
<br><br><br><br><br>
 
<br><br><br><br><br>
Line 210: Line 228:
 
<li>Transformation of 35S-BCR3_Full construct and plating to grow over weekend.</li>
 
<li>Transformation of 35S-BCR3_Full construct and plating to grow over weekend.</li>
 
<li>Transformation of level 1 constructs (35S/GFP/terminors) using both bought and made competent cells to compare - plate out and grown over the weekend at 22℃. </li>
 
<li>Transformation of level 1 constructs (35S/GFP/terminors) using both bought and made competent cells to compare - plate out and grown over the weekend at 22℃. </li>
−
<li>Colony counting for Interlab study completed and Submitted to iGEM HQ.</li>
+
<li>Colony counting for Interlab study completed and Submitted to iGEM HQ. (RC and EM)</li>
 
<li>More seeds of Nicotiana benthamiana were planted and the condition of existing plants was monitored.</li>
 
<li>More seeds of Nicotiana benthamiana were planted and the condition of existing plants was monitored.</li>
 +
<li> GUS and ATH level 0 dig/lig (RC and AT). </li>
 
</ul>
 
</ul>
 
<br><br><br><br><br>
 
<br><br><br><br><br>
Line 222: Line 241:
 
<li>Colony PCR - Lvl 1 constructs with different terminators - Ran on a gel-didn’t get bands.</li>
 
<li>Colony PCR - Lvl 1 constructs with different terminators - Ran on a gel-didn’t get bands.</li>
 
<li>Set up more colony PCRs for the white colonies overnight.</li>
 
<li>Set up more colony PCRs for the white colonies overnight.</li>
 +
<li> All GUS and ATH lvl 0 cells are blue (RC and AT) </li>
 +
<li> Set up level 1 dig/lig reaction with RTBV, Enh, GFP, and NosT (REGT) and liagted overnight (RC and AT)</li>
 
</ul>
 
</ul>
 
<br><br><br><br><br>
 
<br><br><br><br><br>
Line 232: Line 253:
 
<li>PCR on more white colonies from the Lvl 1 plates (35s long/GFP/terminators). </li>
 
<li>PCR on more white colonies from the Lvl 1 plates (35s long/GFP/terminators). </li>
 
<li>Ran the PCR products on a gel - no bands.</li>
 
<li>Ran the PCR products on a gel - no bands.</li>
 +
<li> Transformed cells with REGT (RC and AT). </li>
 
</ul>
 
</ul>
 
<br><br><br><br><br>
 
<br><br><br><br><br>

Revision as of 13:41, 30 September 2018


09/07/2018

  • First day in the lab
  • Had safety induction and signed appropriate forms (all)
  • Discuss medal criteria and how the project fits in with this (all)
  • Discuss overall project and specific techniques (all)
  • Make LB broth (all)
  • Produce cultures to grow overnight at 37℃, A2 and 18J (all)






10/07/2018

  • Miniprep the colonies grown overnight (all)
  • Colony PCRs on Lvl 0 plates 1-9.
  • 1= WRKY intron/ 2= SP3.5.5/ 3= SP3.5.3/ 4= BCR3.5.5/ 5= BCR3.5.3/ 6= BCR3.FULL.5/ 7= BCR3.FULL.3/ 8= ATHSPR1P/ 9= RTBVP






11/07/2018

  • PCR Products from yesterday run on an agarose gel.
  • Set up Lvl 0 reactions for the ones that didn’t have good results from the gel
  • Digestion, Ligation and transformation (SP3.5.3/ BCR3.FULL.3/ ATHSPR1P and 10= GUS).
  • Pick new colonies from plates 1,4 and 5 to redo colony PCR, then run on a gel.
  • Regrew the colonies from plates 2,6 and 9 overnight at 37℃.
  • Make competent E. coli cells. Do a transformation with 18J to test them and plate them out, grow them overnight at 37℃.
  • Prepare for the UK meetup- poster/presentation/worksheets for the workshops.






12/07/2018

  • Half the team in oxford for the UK meet-up (RC, EH, ST and EM)
  • Run gel from colony PCRs from yesterday (HE and AT)
  • Miniprep the colonies from plates 2,6 and 9 (HE and AT)
  • Colony PCRs using the Lvl 0 colonies (3,7,8 and 10) and then run on a gel (HE and AT)
  • Fresh colonies from plates 1,4 and 5 to do colony PCRs and run on a gel (HE and AT)
  • Perform PCR on miniprepped colonies from plates 2,6 and 9 and run on a gel (HE and AT)
  • Regrew colonies from plates 7, 4,5 and 3 overnight at 37℃ (HE and AT)






13/07/2018

  • Half the team in oxford for the UK meet-up (RC, EH, ST and EM)
  • Miniprepped cultures grown overnight from plates 3,4,5 and 7 (HE and AT)
  • PCR on the minipreps from 3,4,5 and 7 (HE and AT)
  • Colony PCR on colonies from plates 1,8 and 10 (HE and AT)
  • Run PCR products on a gel (HE and AT)
  • Picked 2 colonies from plate 10 that worked and grew them over the weekend (HE and AT)






16/07/2018

  • Miniprepped the WRKY intron cultures grown over the weekend.
  • Make plates containing Chloro/XGAL/IPTG and Kan/XGAL/IPTG.
  • iGEM white check in form for using aphids (Health and Safety).
  • Email Nottingham iGEM team about collaborations.
  • Discuss with water institute about helping other teams for collaborations.
  • Email Bayer for human outreach aspect.
  • Set up Lvl 0 reactions for GUS, ATH, and enhancer (digestion/ligation/transformation). (RC and AT)
  • Transform enhancer from biobrick plate 6 L14.
  • PCR all of the miniprepped Lvl 0 constructs to check that they are right- run on a gel.
  • Sent off Lvl 0 constructs for sequencing.
  • Grew up more colonies from previous colonies that contain correct inserts.






17/07/2018

  • Miniprepped colonies grown overnight.
  • Set up Lvl 1 reactions for successful Level 0 constructs.
  • Contacted Thea (European iGEM ambassador) about collaborations.
  • Colony PCR for Lvl 0 colonies (Enhancer, 7 and 10).
  • Run PCR products on a gel - there were no bands.
  • Discussion about logo design.
  • GUS, ATH and Enhancer didn't work, so re-performed Dig/Lig (RC and AT).






18/07/2018

  • Transformation of bacteria for Lvl 1 reactions.
  • Contact Fiona about risk assessment for human outreach event at Techniquest.
  • Meeting with Dan to discuss 3D printing for human outreach.
  • Transformations using the iGEM registry parts for the interlab study.
  • GUS,ATH, Enh cells grew but were all white. Performed colony PCR (cPCR) anyway but none worked. (RC and AT).






19/07/2018

  • Colony PCR for new lvl 1 plates with primers 64+69 and gel run.
  • Colonies with correct band sizes grown overnight.
  • Aphids obtained and placed onto tobacco plant.
  • Dig/Lig on GUS, ATH and Enh again. Transformed and grew overnight (RC and AT).
  • Made new plates with surface IPTG (40 microlitres of 100mM) and X-Gal (96microlitres of 25mg/ml) (RC and AT)
  • Transformed InterLab study constructs (RC, AT and EM)






20/07/2018

  • Contacted Alice about human outreach event in North Wales.
  • Miniprepped level 1 colonies grown overnight, then tested again with primers 39+69.
  • Colony PCR for level 1 plates that didn’t show bands in yesterday’s colony PCR.
  • More seeds of Nicotiana benthamiana were planted and the condition of existing plants was monitored.
  • ATH and GUS were pale blue so left in fridge over weekend (RC and AT).
  • Dig/Lig again on GUS, ATH and Enhancer. Transformed these and redid 2N and 2F interlab plates due to poor growth, allowed to grow over weekend (RC and AT).
  • Picked 2 colonies from each InterLab plate and grew over weekend (RC, AT and EM).






23/07/2018

  • Colony PCR for level 1 plates that haven’t shown bands yet.
  • PCR for Samples 13, 14, 15, 16a, 16b with 39 + 69 primers, ran gel - No bands except those in all samples - discussed possibility of water contamination/contamination in primers.
  • Made new plates and re-streaked colonies that haven’t yet worked from level 1 plates.
  • Grow overnight culture for the competent cells.
  • Level 0 digest for ATH and GUS.
  • Contacted iGEM HQ about collaborations.
  • Tutorial on how to use plate reader for interlab study
  • cPCR on InterLab plates (RC, AT, AND EM)







24/07/2018

  • Colony PCR for new streaked level 1 plates.
  • Re-run PCR for successful level 1 minipreps with new diluted primers 39+69.
  • Re-test level 1 colonies with primers 64+69 to double check before sending for sequencing.
  • Re-grew successful level 1 colonies.
  • Grow up overnight culture for competent cells in 800ml LB - cells didn’t grow properly so couldn't continue with competent cell protocol - instead set up a new overnight culture for the competent cells to start again tomorrow.
  • Transform G7 and 35S terminators from the iGEM registry (plate 6 14N + 16J) and then plate out and grow overnight.
  • 1st Calibration completed for Interlab study, performed at 37°C.
  • Enhancer level 0 worked but GUS and ATH did not. Picked and grew enhancer overnight with good InterLab cells (RC and AT).
  • Set up level 0 dig/lig with GUS and ATH (RC and AT)






25/07/2018

  • Ran gel for yesterday’s PCRs.
  • Miniprepped colonies grown overnight and sent off for sequencing.
  • Tested all colonies from level 1 plates that haven’t yet worked with primers 64+69 and ran gel.
  • Make competent cells from the overnight culture.
  • Test the competent cells by doing a transformation using 18J, then plate out and grow overnight.
  • Colony PCR using the 14N and 16J colonies.
  • Run PCR products on a gel.
  • Grow up 14N and 16J colonies overnight.
  • Grow up GFP and 35S long from the glycerol stocks overnight.
  • 2nd and 3rd Calibration completed, performed at 37°C.
  • Entered ILS data into excel (RC).
  • Performed culture part of ILS, took and measured t0 samples at 10:55, then t6 at 4:55. Measured Abs and Fluorescence (RC and EM).
  • Miniprepped enhancer (HE).






26/07/2018

  • Re-do level 1 reaction for 35S-BCR3_Full construct that has not yet worked - digestion and ligation overnight.
  • Miniprepped 14N, 16J, GFP and 35S from the overnight cultures.
  • Level 1 digest and overnight ligation using 35S/GFP/different terminators (Nos/G7/35S terminator).
  • Level 1 digest and overnight ligation using RTBVp/Enhancer/GFP/Nos.
  • Cell measurement protocol continued, dilution and measurements taken.
  • Colonies incubated overnight for Interlab study CFU section (RC and EM).
  • Entered ILS data and submitted measurement part (RC)






27/07/2018

  • Transformation of 35S-BCR3_Full construct and plating to grow over weekend.
  • Transformation of level 1 constructs (35S/GFP/terminors) using both bought and made competent cells to compare - plate out and grown over the weekend at 22℃.
  • Colony counting for Interlab study completed and Submitted to iGEM HQ. (RC and EM)
  • More seeds of Nicotiana benthamiana were planted and the condition of existing plants was monitored.
  • GUS and ATH level 0 dig/lig (RC and AT).






30/07/2018

  • Colony PCR - plates 1,2,3,4,5,6
  • Colony PCR - Lvl 1 constructs with different terminators - Ran on a gel-didn’t get bands.
  • Set up more colony PCRs for the white colonies overnight.
  • All GUS and ATH lvl 0 cells are blue (RC and AT)
  • Set up level 1 dig/lig reaction with RTBV, Enh, GFP, and NosT (REGT) and liagted overnight (RC and AT)






31/07/2018

  • Ran PCRs from yesterday on a gel - one colony had a band of the expected size so picked and grew that colony overnight.
  • PCR on more white colonies from the Lvl 1 plates (35s long/GFP/terminators).
  • Ran the PCR products on a gel - no bands.
  • Transformed cells with REGT (RC and AT).






01/08/2018

  • Level 1 transformation.
  • Grow up the 35s long and GFP overnight.
  • Miniprepped the 35S/GFP/35Sterminator colony that was grown overnight.
  • Miniprepped level 1 cultures grown overnight (14 and 16).
  • PCR on the miniprepped DNA (35S/GFP/35Sterminator and 14 and 16), the PCR products were then run on a gel.






02/08/2018

  • Sent for sequencing - 35SLong-GFP-35STerm with primer 69 and 35S-BCR3 with primer 64.
  • Blue/white selection on new transformed plates was poor so left in fridge overnight.
  • Miniprepped the 35S long and GFP grown overnight.
  • Set up new level 1 reactions for GFP with the various terminators.
  • Transformations using the level 1 reactions - plated onto Kan/IPTG and XGAL and grown overnight at 37℃.






03/08/2018

  • Sent 35S-BCR3 for sequencing with primer 69.
  • Colony PCR for level 1 plates.
  • More seeds of Nicotiana benthamiana were planted and the condition of existing plants was monitored.
  • Colony PCR on the level 1 plates (GFP with various terminators) from yesterday - ran on a gel - one colony had a band of the expected size so that was picked and grown over the weekend.
  • Some of the plates didn’t have many colonies on so replated the rest of the transformation mixture from yesterday.






06/08/2018

  • Grew up all level 0 parts overnight.






07/08/2018

  • Did level 1 reaction for 1-7 plates with NEB kit.
  • Miniprepped 35S long/GFP/G7 construct looked good so was sent for sequencing.
  • Redid the level 1 reactions (35S long/GFP/various terminators) with a new protocol.
  • Transformed the level 1 reactions and plated out on Kan/IPTG/XGAL and grew overnight at 37℃.






08/08/2018

  • Ran colony PCR on new level 1 plates - gel wasn’t great (EH)
  • PCR run with 14,15,16,18, primers 64+70 and 64+71 were used - bands for 64+71 were strong (ST and HE)
  • Initial contact with WashU about potential collaboration via email and instagram (LT and EM)
  • Colony PCR on white colonies from the plates grown overnight. Ran the PCR products on a gel. Two colonies had bands around the expected size so were picked and grown overnight (EH)
  • Replated the rest of the level 1 transformation mix onto new plates (EH)






09/08/2018

  • Repeated colony PCR on new level 1 plates - gel slightly better so put successful colonies to grow overnight (EH)
  • Made new chloro broth (EH)
  • Grew up level 0 parts in correct broth (EH)
  • PCR run with 14,15,16,18, primers 69+70 and 69+71 were used. Bands for 69+70 were strong (ST and HE)
  • Video call with WashU detailing possible collaboration (all)
  • Miniprepped the two colonies grown overnight (EH)
  • Colony PCR on the white colonies from the level 1 plates (35S/GFP/terminators) and PCR on the miniprepped colonies (EH)
  • Ran the PCR products on the gel- only the miniprepped construct containing the G7 terminator had a band of the expected size (EH)






10/08/2018

  • Sent the level 1 construct containing 35S long/GFP/G7 off for sequencing with primer 69 (EH)
  • Picked more white colonies off the terminator level 1 plates for PCR (EH)
  • Ran the PCR products on a gel - there were no bands of the expected size (EH)






13/08/2018

  • Sent the 35S long promoter off for sequencing with primer 57 (EH)
  • Set up a level 1 reaction using 35S short/Enhancer/GFP/Terminators (EH)
  • Transformation of the level 1 reaction - plate out onto Kan/IPTG/XGAL (EH)
  • Regrow the 35S long overnight (EH)






14/08/2018

  • Colony PCR on the white colonies from the level 1 reactions from yesterday (EH)
  • Run PCR products on a gel - most of the bands look good (EH)
  • Pick and grow the colonies overnight which had bands of the expected size on the gel (EH)
  • Miniprepped the 35S long which was grown overnight (EH)
  • Further discussion on logo design (EM and RC)






15/08/2018

  • Sent 35S long for sequencing with primer 68 (EH)
  • Miniprepped the cultures grown overnight (EH)
  • Sent one of each level 1 construct with different terminators off for sequencing (35S/enhancer/GFP/terminators) (EH)
  • Agro transformation with the level 1 constructs containing GFP and different terminators (EH)
  • Plants monitored and watered (EM)






16/08/2018

  • Grow seed overnight for making competent cells (EH)
  • Made up a solution of MgCl2 and autoclaved it (EH)
  • Obtaining contact information for potential collaborations (EM)






17/08/2018

  • Made competent E. coli (EH)
  • Transform new competent cells with 18J to test them - plate onto chloramphenicol and grow on the bench over the weekend. (EH)
  • Streaked out the agro plates and grow over the weekend at 28℃.
  • Sequencing came back for the 35S long, turned out it was 35S short which explains why the level 1s were not working.






20/08/2018

  • Pick colonies off the agro plates and grow cultures overnight in LB broth, Kan and Rif.






21/08/2018

  • Infiltrated plants with 35S-GUS and either NosT, G7, or 35S (EH)
  • Re-potted plants (EM)






22/08/2018

  • Level 1 Ligation for (15)cRTBVp_BCR3_Full and (18) 35S_BCR3_Full (HE)
  • Transformation of cells with Level 1s (HE)
  • Grew up four 11 35s (HE)






23/08/2018

  • Preparation for Operation Earth. (LT)
  • Checked on progress of re-potted plants. (EM)
  • Miniprep of 35s (HE)
  • RNA Extraction from plants 14 and 16 (+ve and -ve) (ST)
  • Colony PCR for Plates 2 and 5 (HE)






24/08/2018

  • RT Reaction (-ve, 14, 16) (ST)






28/08/2018

  • Tested new primers with 14 and 16 (ST)






29/08/2018

  • PCR with cDNA + gDNA (ST)






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14/09/2018

  • Last official day so cleaned up the lab (all)






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31/10/2018

  • 2spooky4me