Difference between revisions of "Team:Oxford/Notebook"
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<li><a href="#8q4">Day 4</a></li> | <li><a href="#8q4">Day 4</a></li> | ||
<li><a href="#8q5">Day 5</a></li> | <li><a href="#8q5">Day 5</a></li> | ||
| − | </ul> | + | <li><a href="#8q7">Day 7</a></li> |
| + | </ul> | ||
</li> | </li> | ||
<li> | <li> | ||
| Line 469: | Line 470: | ||
<h3>Day 3</h3> | <h3>Day 3</h3> | ||
<p> | <p> | ||
| + | Transformations of fck, ptcg, pmg, tc and cg into DH5-alpha. Results: unsuccessful for all except cg. Chloroamphenicol | ||
| + | </p> | ||
| + | <p> | ||
| + | Transformation of mg(pBAD) into MG1655, Ampicillin, Successful. | ||
| + | </p> | ||
| + | <p> | ||
| + | Digestion and diagnostic gel of M3 (1-6). On gel, expected band size = 287bp. Used 2% agrose gel. | ||
| + | </p> | ||
| + | <p> | ||
| + | PCR m out of mg shipping vector. Expected band size = 196bp Successful Extracted | ||
</p> | </p> | ||
</section> | </section> | ||
| Line 474: | Line 485: | ||
<h3>Day 4</h3> | <h3>Day 4</h3> | ||
<p> | <p> | ||
| + | Miniprep cg | ||
| + | </p> | ||
| + | <p> | ||
| + | Diagnostic gel for cg, use EcoRI and SpeI. Expected band size = 1250bp | ||
| + | </p> | ||
| + | <p> | ||
| + | Plate reader experiment for pg and negative control, followed protocol. 50ul LB (not 40ul) OD 600 | ||
| + | </p> | ||
| + | <p> | ||
| + | Transforms: | ||
| + | Pmg – chlorophenicol into DH5alpha, Fck – chloroamphenicol into DH5alpha, Ptcg – chloroamphenicol into DH5alpha, mEx – amphicillin into DH5alpha, pcg – chloroamphenicol into DH5alpha, pBAD vector no insert = amphicillin MG1655. | ||
</p> | </p> | ||
</section> | </section> | ||
| Line 487: | Line 509: | ||
<h3>Day 1</h3> | <h3>Day 1</h3> | ||
<p> | <p> | ||
| + | PCR ptcg, pmg, cg and fck. For ptcg – use old forward (EcoRI), new reverse (SpeI), 60o annealing. For pmg – use old forward (EcoRI), new reverse (SpeI), 60o annealing. For cg – use old forward (EcoRI), new reverse (SpeI), 60o annealing. For fck – use old forward (EcoRI), new reverse (SpeI), 60o annealing. All 1 min elongation time and ran 2 of each. | ||
| + | Results: ptcg, pmg, cg and fck successful | ||
| + | </p> | ||
| + | <p> | ||
| + | Gel extract all 4 parts and mymT in expression (Mex) | ||
| + | </p> | ||
| + | <p> | ||
| + | Digestion for all 4 parts and mEx using EcoRI and SpeI for ptcg, pmg, cg and fck, XbaI and PstI for mEx | ||
| + | </p> | ||
| + | <p> | ||
| + | Ligation of ptcg, pmg, tc, cg, pcg, mex and fck | ||
| + | </p> | ||
| + | <p> | ||
| + | Nanodrop cg and fck, 7.6 and 18.8, respectively. Start from PCR stage. | ||
| + | </p> | ||
| + | <p> | ||
| + | PCR m out of mg (retry) | ||
| + | 62o annealing – 15s, 72o elongation – 15s | ||
</p> | </p> | ||
</section> | </section> | ||
| Line 492: | Line 532: | ||
<h3>Day 2</h3> | <h3>Day 2</h3> | ||
<p> | <p> | ||
| + | Started using improved cloning procedures to attempt to get everything working | ||
| + | </p> | ||
| + | Starting from PCR with tc, cg, ptcg, pmg, pcg and fck. Old forward, new reverse primers for cg, ptcg, pmg, pcg and fck. Old forward, old reverse primer for tc. | ||
| + | </p> | ||
| + | <p> | ||
| + | Transform Fla-Art175 (part from last year) and tc(shipping) into DH5-alpha, use chloroamphenicol. Fla-Art175 = 1120 bp. tc = 545 bp. PSB1C3C shipping plasmid backbone = 2070bp | ||
| + | </p> | ||
| + | <p> | ||
| + | Transform pmg, Mex and tc (pBAD) into DH5-alpha, use chloroamphenicol for pmg and use ampicillin for mEx and tc. | ||
| + | </p> | ||
| + | <p> | ||
| + | Pick colonies of pBAD (no insert) to make pBAD stock. Pick x8 | ||
</p> | </p> | ||
</section> | </section> | ||
| Line 497: | Line 549: | ||
<h3>Day 3</h3> | <h3>Day 3</h3> | ||
<p> | <p> | ||
| + | Miniprep pBAD inset x6. Ran x2 samples from each picked colony. | ||
| + | Digest with NcoI and PstI. Only digested using sample from 1a. | ||
</p> | </p> | ||
| + | <p> | ||
| + | PCR tc, cg, ptcg, pcg 1, fcg 2. tc - use old forward, old reverse, 62o annealing. cg - use old forward, new reverse, 60o annealing. ptcg - use old forward, new reverse, 62o annealing. pcg 1 - use old forward, new reverse, 60o annealing. fcg 2 - use old forward, new reverse, 62o annealing. | ||
| + | </p> | ||
| + | <p> | ||
| + | Gel extract to make pBAD stock solution. Used 1a colony. Send undigested plasmid for sequencing. Put 1a gel in freezer. Gel extract if sequencing comes back correct. | ||
| + | </p> | ||
| + | <p> | ||
| + | Pick colonies of: Fla-Art175 (x4, chloroamphenicol), Tc (shipping) for vector (x4, chloroamphenicol), mEx (x6, amphicillin), Tc in pBAD (x6, amphicillin). | ||
| + | </p> | ||
| + | <p> | ||
| + | PCR tc out of shipping tc2 using ExP Rev and Exp Tat Csp1 primers, 62o annealing. | ||
| + | Ran gel → got one band on each so can do PCR purification rather than gel extraction</p> | ||
</section> | </section> | ||
<section id="8q4"> | <section id="8q4"> | ||
<h3>Day 4</h3> | <h3>Day 4</h3> | ||
<p> | <p> | ||
| + | Miniprep tc for shipping, Fla-Art175 shipping and mEx. Tc shipping = 1a, 1b, 2a, 2c, Fla-Art175 shipping = 3a, 3b, 4a, 4c, mEx = A, B, C, D, E | ||
</p> | </p> | ||
| + | <p> | ||
| + | Digestion for above parts, 1a to 4b using EcoRI and SpeI. A to E using BamHI and PstI (use 2% gel) | ||
| + | </p> | ||
| + | <p> | ||
| + | Gel for Mex (A to E) came back successful, nanodropped A to E. C had highest value (35.5) → sent for sequencing | ||
| + | </p> | ||
| + | <p> | ||
| + | Gel for 1a-4a, 1a = tc, 2a = tc, 3a = Fla-Art175, 4a = Fla-art175. Kept 1a and 2a. Set up more to digest overnight | ||
| + | </p> | ||
| + | <p> | ||
| + | Pick colonies for mEx and pg (MG1655)</p> | ||
</section> | </section> | ||
<section id="8q5"> | <section id="8q5"> | ||
<h3>Day 5</h3> | <h3>Day 5</h3> | ||
<p> | <p> | ||
| + | Miniprep mEx (1-4), digest, diagnostic gel, sequence. Nandrop results: 1 = 95.8, 2 = 99.4,3 = 89.3, 4 = 93.5 | ||
| + | Discarded 3. | ||
| + | </p> | ||
| + | <p> | ||
| + | Re-transform pg into MG1655 | ||
| + | </p> | ||
| + | <p> | ||
| + | Run mEx gel, Ran mEx 1-4. | ||
| + | Sending mEx2 for sequencing. | ||
| + | Keep mEx1 just in case. | ||
| + | </p> | ||
| + | <section id="8q7" | ||
| + | <h3>Day 7</h3> | ||
| + | <p> | ||
| + | Made more chloramphenicol | ||
| + | </p> | ||
| + | <p> | ||
| + | Pick colonies of fcg, cg, ptcg, fck, pcg, pmg and mEx, 6x of each, apart from pmg (only 1x). | ||
| + | Chloramphenicol for all except Mex (ampicillin) | ||
</p> | </p> | ||
</section> | </section> | ||
Revision as of 08:15, 17 October 2016
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
Day 1
Day 2
Day 3
Day 4
Day 5
Week 2
Day 1
Day 2
Day 3
Day 4
Day 5
Week 3
Day 1
Day 2
Day 3
Day 4
Day 5
Week 4
Day 1
Day 2
Day 3
Day 4
Day 5
Week 5
Day 1
Day 2
Day 3
Day 4
Day 5
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
PCR ptcg and pmg. ptcg = new forward, old reverse primers,60o annealing temperature
Transform pg and mg into MG1655, pg uses chloroamphenicol, mg uses ampicillin.
Week 7
Day 1
Measuring fluorescence in 96 well plate of pg
Transformation of mg(pBAD) into MG1655 using ampicillin.
Pick 4x colonies from pg plate using chloroamphenicol.
Pick 4x colonies from MG1655, no antibiotic.
Ligate ptcg, pmg, tc, cg and fck
Day 2
Miniprep M3 using ampicillin.
Digest and diagnostic gel using EcoRI and SpeI. Expected band size = 254bp Came back incorrect
Pick colonies for pCusC, MG1655 negative control, MG into pBAD using chloroamphenicol, no antibiotic and amphicillin, respectively. 4 colonies each
Transformations of fck, ptcg, pmg, tc and cg into DH5-alpha, chloroamphenicol need 10 plates. Not successful.
Day 3
Transformations of fck, ptcg, pmg, tc and cg into DH5-alpha. Results: unsuccessful for all except cg. Chloroamphenicol
Transformation of mg(pBAD) into MG1655, Ampicillin, Successful.
Digestion and diagnostic gel of M3 (1-6). On gel, expected band size = 287bp. Used 2% agrose gel.
PCR m out of mg shipping vector. Expected band size = 196bp Successful Extracted
Day 4
Miniprep cg
Diagnostic gel for cg, use EcoRI and SpeI. Expected band size = 1250bp
Plate reader experiment for pg and negative control, followed protocol. 50ul LB (not 40ul) OD 600
Transforms: Pmg – chlorophenicol into DH5alpha, Fck – chloroamphenicol into DH5alpha, Ptcg – chloroamphenicol into DH5alpha, mEx – amphicillin into DH5alpha, pcg – chloroamphenicol into DH5alpha, pBAD vector no insert = amphicillin MG1655.
Day 5
Week 8
Day 1
PCR ptcg, pmg, cg and fck. For ptcg – use old forward (EcoRI), new reverse (SpeI), 60o annealing. For pmg – use old forward (EcoRI), new reverse (SpeI), 60o annealing. For cg – use old forward (EcoRI), new reverse (SpeI), 60o annealing. For fck – use old forward (EcoRI), new reverse (SpeI), 60o annealing. All 1 min elongation time and ran 2 of each. Results: ptcg, pmg, cg and fck successful
Gel extract all 4 parts and mymT in expression (Mex)
Digestion for all 4 parts and mEx using EcoRI and SpeI for ptcg, pmg, cg and fck, XbaI and PstI for mEx
Ligation of ptcg, pmg, tc, cg, pcg, mex and fck
Nanodrop cg and fck, 7.6 and 18.8, respectively. Start from PCR stage.
PCR m out of mg (retry) 62o annealing – 15s, 72o elongation – 15s
Day 2
Started using improved cloning procedures to attempt to get everything working
Starting from PCR with tc, cg, ptcg, pmg, pcg and fck. Old forward, new reverse primers for cg, ptcg, pmg, pcg and fck. Old forward, old reverse primer for tc.Transform Fla-Art175 (part from last year) and tc(shipping) into DH5-alpha, use chloroamphenicol. Fla-Art175 = 1120 bp. tc = 545 bp. PSB1C3C shipping plasmid backbone = 2070bp
Transform pmg, Mex and tc (pBAD) into DH5-alpha, use chloroamphenicol for pmg and use ampicillin for mEx and tc.
Pick colonies of pBAD (no insert) to make pBAD stock. Pick x8
Day 3
Miniprep pBAD inset x6. Ran x2 samples from each picked colony. Digest with NcoI and PstI. Only digested using sample from 1a.
PCR tc, cg, ptcg, pcg 1, fcg 2. tc - use old forward, old reverse, 62o annealing. cg - use old forward, new reverse, 60o annealing. ptcg - use old forward, new reverse, 62o annealing. pcg 1 - use old forward, new reverse, 60o annealing. fcg 2 - use old forward, new reverse, 62o annealing.
Gel extract to make pBAD stock solution. Used 1a colony. Send undigested plasmid for sequencing. Put 1a gel in freezer. Gel extract if sequencing comes back correct.
Pick colonies of: Fla-Art175 (x4, chloroamphenicol), Tc (shipping) for vector (x4, chloroamphenicol), mEx (x6, amphicillin), Tc in pBAD (x6, amphicillin).
PCR tc out of shipping tc2 using ExP Rev and Exp Tat Csp1 primers, 62o annealing. Ran gel → got one band on each so can do PCR purification rather than gel extraction
Day 4
Miniprep tc for shipping, Fla-Art175 shipping and mEx. Tc shipping = 1a, 1b, 2a, 2c, Fla-Art175 shipping = 3a, 3b, 4a, 4c, mEx = A, B, C, D, E
Digestion for above parts, 1a to 4b using EcoRI and SpeI. A to E using BamHI and PstI (use 2% gel)
Gel for Mex (A to E) came back successful, nanodropped A to E. C had highest value (35.5) → sent for sequencing
Gel for 1a-4a, 1a = tc, 2a = tc, 3a = Fla-Art175, 4a = Fla-art175. Kept 1a and 2a. Set up more to digest overnight
Pick colonies for mEx and pg (MG1655)
Day 5
Miniprep mEx (1-4), digest, diagnostic gel, sequence. Nandrop results: 1 = 95.8, 2 = 99.4,3 = 89.3, 4 = 93.5 Discarded 3.
Re-transform pg into MG1655
Run mEx gel, Ran mEx 1-4. Sending mEx2 for sequencing. Keep mEx1 just in case.
Made more chloramphenicol
Pick colonies of fcg, cg, ptcg, fck, pcg, pmg and mEx, 6x of each, apart from pmg (only 1x). Chloramphenicol for all except Mex (ampicillin)
Week 9
Day 1
Day 2
Day 3
Day 4
Day 5
Week 10
Day 1
Day 2
Day 3
Day 4
Day 5
Week 11
Day 1
Day 2
Day 3
Day 4
Day 5
Week 12
Day 1
Day 2
Day 3
Day 4
Day 5
Week 13
Day 1
Day 2
Day 3
Day 4
Day 5
Week 14
Day 1
Day 2
Day 3
Day 4
Day 5
Week 15
Day 1
Day 2
Day 3
Day 4
Day 5
Week 16
Day 1
Day 2
Day 3
Day 4
Day 5
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
