Team:Oxford/Notebook
Notebook
Introduction
This page documents all the experiments we carried out in the wet lab as a part of our project. The details of the process we carried out can be found on our protocols page, and the chemicals we used can be found on the chemicals page. The interlab project was carried out alongside the rest of the wet lab but we have recorded it separately.
Week 1
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Week 2
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Week 3
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Week 4
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Week 5
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Week 6
Day 1
Transform ptcg and pmg 2 plates each, therefore 4 plates
Feedback pCusC mKATE (fck) arrived 1000ng delivered → add 100ul to give 10ng/ul After resuspension, dilute 1 in 10 to give 1ng/ul
PCR fck fck,on 60 = old forward, old reverse primers, 60p annealing fck,on 62 = old forward, old reverse primers, 62o annealing fck,on 60 = old forward, new reverse primers, 60o annealing fck, on 62 = old forward, new reverse primers, 62o annealing Results: fck on 60 and fck on 62 worked
Gel extraction for fck
Day 2
Picked colonies from pmg and ptcg
Day 3
Miniprep ptcg, pmg and pm
Diagnostic gel for ptcg and pmg Using PstI and EcoRI Expected band sizes = 1824 and 1565, respectively
Sent ptcg and pmg for sequencing
Day 4
Sequencing results ptcg → didn’t work pmg → didn’t work cg (in shipping plasmid) → didn’t work tc → didn’t work pg → has promoter, no mismatches, truncated mg → good
Next steps Re-transform fck PCR ptcg, pmg, cg and tc Pick M3 colonies Transform pg and mg into MG1655
Old shipping plasmid possibly contaminated, digest new. Using part from last year (Art-175). Component: Art-175 5ul, 10xbuffer 5ul, EcoRI 1ul, SpeI 1ul and MilliQ 38ul. Set up digest at 37o for 2 hours
Next steps: Heat inactivate at 80o for 10 mins. Dephosphorylate with 1ul antarctic phosphatase 30 mins at 37o. Run gel. Excise 2kb band.
PCR tc, cg, ptcg and pmg. Tc – 60o annealing, old forward, old reverse. Cg – 62o annealing, old forward, new ptcg reverse. Ptcg – 60o annealing, new forward, old reverse. Pmg – 60o, new forward, old reverse.
Gel for tc, cg, ptcg and pmg Expected band size = 545, 1277, 1824 and 1565 bp, respectively Results: tc = very smeary, other 3 to be extracted
Day 5
Week 7
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Week 8
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Week 9
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Week 10
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Week 11
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Week 12
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Week 13
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Week 14
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Week 15
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Week 16
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Interlab
Day 1
Transformed the 5 interlab parts into DH5a as per the transformation protocol.
Day 2
Transformations mostly unsuccessful. Started making new competent cells.
Day 3
Second day of competent cell preparation, cells frozen and put in the freezer.
Day 4
LB media preparation and re-transformation of the interlab parts.
Day 5
Had colonies from all interlab parts except PC. Picked colonies from all successful plates. Retransformed PC.
Day 6
PC didn't transform again. After measuring the sample we were sent with nanodrop we found the sample has no DNA in it. Requested a second sample from iGEM HQ Nevertheless we did a third transformation for PC. NC and TD1-3 were miniprepped and sent for sequencing.
Day 7
TD1 sequencing successful, others had the wrong part in so retransformed these. PC colonies successful despite nanodrop so picked colonies for these.
Day 8
PC miniprepped, digested and sent for sequencing. Colonies picked for re-transformed TD2, TD3 and NC.
Day 9
PC sequencing unsuccessful. TD2, TD3 and NC miniprepped and sent for sequencing.
Day 10
TD2 sequencing successful, others not. PC, NC and TD3 transformed again
Day 11
PC, NC and TD3 colonies picked.
Day 12
PC, NC and TD3 miniprepped and sent for sequencing.
Day 13
NC and TD3 sequencing successful but PC wrong so retransformed again.
Day 14
Picked colonies for PC.
Day 15
PC was miniprepped and digested. Gel showed it contained the wrong part so we transformed the part from the distribution kit instead.
Day 16
Colonies picked for PC.
Day 17
PC miniprepped, digested and sent for sequencing. Produced more competent cells.
Day 18
PC sequencing correct! All 5 interlab parts were transformed into MG1655
Day 19
Made the calibration curves for the OD600 and the FITC standard curve Colonies picked for all the interlab parts.
Day 20
Tested iGEM's protocol for the OD600 and the fluorescence. Picked more colonies for all 5 parts.
Day 21
Repeated iGEM's protocol and started Oxford iGEM's protocol.
Day 22
Finished Oxford iGEM's protocol and submitted the data obtained using iGEM's method to iGEM
