Difference between revisions of "Team:Cardiff Wales/Notebook/Text"
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<li>Did level 1 reaction for 1-7 plates with NEB kit.</li> | <li>Did level 1 reaction for 1-7 plates with NEB kit.</li> | ||
<li>Miniprepped 35S long/GFP/G7 construct looked good so was sent for sequencing. </li> | <li>Miniprepped 35S long/GFP/G7 construct looked good so was sent for sequencing. </li> | ||
Revision as of 20:24, 19 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 and 8 (digestion/ligation/transformation).
- 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.
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.
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.
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.
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
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.
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
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.
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.
- More seeds of Nicotiana benthamiana were planted and the condition of existing plants was monitored.
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.
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.
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)
30/08/2018
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14/09/2018
- Last official day so cleaned up the lab (all)
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31/10/2018
- 2spooky4me