Team:NTNU Trondheim/Notebook
From 2014.igem.org
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+ | <div id="week39entry" class="nb-week"> | ||
+ | <div class="twelve columns"> | ||
+ | <h3 class="centered">Week 39</h3> | ||
+ | <h5 class="centered">(22/09 - 28/09)</h5> | ||
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+ | </p> | ||
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+ | <div class="entry nb-wet"> | ||
+ | <div> | ||
+ | <h6>September 22nd | ||
+ | <div class="nb-onetech-i nohilite">show technical details</div> | ||
+ | </h6> | ||
+ | <div class="nb-tech">{{{techdetail}}}</div> | ||
+ | <p> <ol> | ||
+ | <li>Two colonies were picked from LF1, RF1, Kan1, GOx, Syn, LF2+Kan2 and used for colony PCR. Colonies of Kan1 or LF2+Kan2 were inoculated in 4 ml SOC containing 1µg/ml chloramphenicol and kanamycin, while the remaining samples were inoculated in 4 ml SOC containing 1µg/ml chloramphenicol. PCR-products were verified with gel electrophoresis.</li> | ||
+ | </ol> | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
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+ | |||
+ | <div class="entry nb-wet"> | ||
+ | <div> | ||
+ | <h6>September 23rd | ||
+ | <div class="nb-onetech-i nohilite">show technical details</div> | ||
+ | </h6> | ||
+ | <div class="nb-tech">{{{techdetail}}}</div> | ||
+ | <p> <ol> | ||
+ | <li>Mini-prep of LF1, RF2, Kan1, GOx1, Syn1, LF+Kan1. Digest of 2x LF2+Kan2 with <i>EcoRI</i> and <i>SpeI</i>. Digests was cleaned with PCR cleanup, and ligated overnight with RF2 and pSB1C3 using T4-ligase.</li> | ||
+ | </ol> | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
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+ | <div class="entry nb-wet"> | ||
+ | <div> | ||
+ | <h6>September 24th | ||
+ | <div class="nb-onetech-i nohilite">show technical details</div> | ||
+ | </h6> | ||
+ | <div class="nb-tech">{{{techdetail}}}</div> | ||
+ | <p> <ol> | ||
+ | <li>Transformed 2x of pSB1C3, LF, Kan and RF into <i>E. coli</i> DH5α. Incubated at 37⁰C overnight.</li> | ||
+ | </ol> | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
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Revision as of 15:27, 14 October 2014
Team:Cornell/notebook
From 2013.igem.org
Notebook
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Week 23
(02/06 - 08/06)
June 4th
show technical details
Made LB plates with ampicillin and ampicillin + kanamycin.
June 6th
show technical details
Made competent E. coli DH5α cells.
Week 24
(09/06 - 15/06)
June 10th
show technical details
Test of heat-shock transformation efficiency of competent E. coli DH5α from June 6th.
June 11th
show technical details
Rehydrated BioBrick BBa_J23101, BBa_B0034 and BBa_C0012, and heat-shock transformed them into competent E. coli DH5α cells, and incubated the cultures on LB plates with ampicillin overnight.
June 12th
show technical details
Inoculated BioBrick colonies from June 11th in SOC medium containing ampicillin.
June 13th
show technical details
Negative control of non-transformed E. coli DH5α on LB plates with ampicillin.
Week 25
(16/06 - 22/06)
June 16th
show technical details
- Negative control of non-transformed E. coli DH5α on LB plates with ampicillin + kanamycin.
- Made new LB plates with ampicillin.
- Heat-shock transformed E. coli DH5α with BioBricks BBa_J23101, BBa_B0034 and BBa_C0012 and plated onto LB with ampicillin.
June 17th
show technical details
- PCR amplification of Left_flank, Righ_flank and Kanamycin_resistance with Synechocystis PCC. δslr0906 as template using touchdown PCR.
- Gel electrophoresis verification of PCR products on agarose gel
June 18th
show technical details
- Mini-prep of PCR products from June 17th.
- PCR amplification of mCherry gene, BioBricks BBa_J23101, BBa_B0034 and BBa_C0012 using touchdown PCR.
- Gel electrophoresis verification of PCR products on agarose gel
June 19th
show technical details
- Digest of BioBricks BBa_J23101, BBa_B0034 and BBa_C0012.
- Gel electrophoresis verification of digest on agarose gel.
June 20th
show technical details
- Made LB plates with chloramphenicol.
- Digest of BBa_J23101, BBa_B0034 and psB1C3 backbone according to the 3A assembly method.
Week 26
(23/06 - 29/06)
June 23rd
show technical details
Ligation and heat-shock transformation of digestion mixtures from June 20th.
June 24th
show technical details
The gel verification of digest showed an unexpected band of the J BioBrick, and based on failed ligation attempts, it was decided to inoculate new BioBrick colonies.
- Attempted ligation and heat-shock transformation of the digestion mixtures from June 20th again.
- Gel electrophoresis verification of digest from June 20th on agarose gel.
- Inoculated new colonies of BBa_J23101, BBa_B0034 and BBa_C0012.
June 25th
show technical details
Verification of Left_flank, Right_flank and Kanamycin_resistance on gel showed expected bands at 566 bp, 552 bp and 961 bp respectively.
- Mini-prep of BioBrick colonies from June 24th.
- Enzymatic digestion of newly mini-prepped BBa_J23101, BBa_B0034 and psB1C3 backbone.
- New PCR amplification of Left_flank, Righ_flank and Kanamycin_resistance using touchdown PCR.
- Gel electrophoresis verification of PCR products on agarose gel.
June 26th
show technical details
The ligation procedure was performed as June 23rd with a few exceptions: (1) increased the volume of ligation mixture, and hence the amount of ligase. This was because it was assumed that the residual activity of PstI-HF could be counteracted by increasing Taq ligase concentration; and (2) created a short time series for ligation, one sample at 42 °C for 20 minutes and another one for one hour. Both ligation times resulted in growth on LB plates with chloramphenicol.
- Gel electrophoresis verification of BBa_J23101, BBa_B0034 and psB1C3 backbone backbone digest.
- Another attempted ligation of digested B, J and psB1C3 backbone from June 25th, and heat-shock transformation into competent E. coli DH5α cells.
June 27th
show technical details
- QIAquick PCR purification of Left_flank, Righ_flank and Kanamycin_resistance PCR product from June 25th.
- Digested and ligated Right_Flank, Kanamycin_resistance and psB1A3 backbone, then heat-shock transformed it into competent E. coli DH5α cells.
- Inoculated colonies from ligation of BBa_J23101, BBa_B0034 and psB1C3 backbone from June 26th.
June 28th
show technical details
Mini-prep of inoculated colonies from BBa_J23101, BBa_B0034 and psB1C3 backbone ligation on June 26th.
Week 27
(30/06 - 06/07)
June 30th
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- Gel electrophoresis verification of BBa_J23101, BBa_B0034 and psB1C3 backbone ligation on agarose gel.
- Digestion, ligation and heat-shock transformation of the ligated JB with Left_flank and psB1A3 backbone.
July 1st
show technical details
The agarose gel of NotI-HF digested {RF, Kan, psB1A3} and {J,B, psB1A3} showed bands at around 1000 bp for all samples, which is wrong in both cases.
- New gel electrophoresis verification of BBa_J23101, BBa_B0034 and psB1C3 backbone ligation on agarose gel using NotI-HF restriction enzyme.
- Also verified Righ_flank, Kanamycin_resistance and psB1A3 backbone ligation from June 27th on agarose gel using NotI-HF.
- Inoculation of Left_flank, JB and psB1A3 backbone colonies from June 30th.
July 2nd
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- Mini-prep of {LF, JB, psB1A3} from June 30th.
- Gel electrophoresis verification of {RF, Kan, psB1A3}, {J,B, psB1A3} and {Left_flank, JB, psB1A3} on agarose gel using NotI-HF restriction enzyme.
- Inoculated new colonies from plates containing colonies from the original BBa_J23101 and BBa_B0034 BioBricks.
July 3rd
show technical details
- Mini-prep of inoculated BBa_J23101 and BBa_B0034 colonies from July 2nd.
- Gel electrophoresis verification of J and B on an agarose gel using NotI-HF restriction enzyme.
- Digested J, B, Left_flank, Righ_flank, Kanamycin_resistance, psB1A3 backbone and psB1C3 backbone.
Week 39
(22/09 - 28/09)
September 22nd
show technical details
- Two colonies were picked from LF1, RF1, Kan1, GOx, Syn, LF2+Kan2 and used for colony PCR. Colonies of Kan1 or LF2+Kan2 were inoculated in 4 ml SOC containing 1µg/ml chloramphenicol and kanamycin, while the remaining samples were inoculated in 4 ml SOC containing 1µg/ml chloramphenicol. PCR-products were verified with gel electrophoresis.
September 23rd
show technical details
- Mini-prep of LF1, RF2, Kan1, GOx1, Syn1, LF+Kan1. Digest of 2x LF2+Kan2 with EcoRI and SpeI. Digests was cleaned with PCR cleanup, and ligated overnight with RF2 and pSB1C3 using T4-ligase.
September 24th
show technical details
- Transformed 2x of pSB1C3, LF, Kan and RF into E. coli DH5α. Incubated at 37⁰C overnight.
Week 40
(29/09 - 05/10)
October 1st
show technical details
- Concentration of miniprepped biobricks LF (24.8 ng/µl), RF (33.3 ng/µl), Kan (40.9 ng/µl), GOx (30.1 ng/µl), Lac (34.6 ng/µl) and Kan + RF (39.8 ng/µl) were measured with nanodrop.