Team:NTNU Trondheim/Notebook
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Team:Cornell/notebook
From 2013.igem.org
Notebook
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Week 23
(02/06 - 08/06)
June 4th
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Made LB plates with ampicillin and ampicillin + kanamycin.
June 6th
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Made competent E. coli DH5α cells.
Week 24
(09/06 - 15/06)
June 10th
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Test of heat-shock transformation efficiency of competent E. coli DH5α from June 6th.
June 11th
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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
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Inoculated BioBrick colonies from June 11th in SOC medium containing ampicillin.
June 13th
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Negative control of non-transformed E. coli DH5α on LB plates with ampicillin.
Week 25
(16/06 - 22/06)
June 16th
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- 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
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- 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
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- 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
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- Digest of BioBricks BBa_J23101, BBa_B0034 and BBa_C0012.
- Gel electrophoresis verification of digest on agarose gel.
June 20th
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- 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
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Ligation and heat-shock transformation of digestion mixtures from June 20th.
June 24th
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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
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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
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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
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- 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
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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
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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
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- 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
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- 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
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- 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
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- Transformed 2x of pSB1C3, LF, Kan and RF into E. coli DH5α. Incubated at 37⁰C overnight.
Week 40
(29/09 - 05/10)
October 1st
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- 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.