Team:NTNU Trondheim/Notebook
From 2014.igem.org
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<li class="has-flyout pg-outreach pg-outreach_presentations pg-outreach_panels pg-outreach_awareness"> | <li class="has-flyout pg-outreach pg-outreach_presentations pg-outreach_panels pg-outreach_awareness"> | ||
- | <a href="https://2014.igem.org/Team:NTNU_Trondheim/Sponsors">Acknowledgements</a> | + | <a href="https://2014.igem.org/Team:NTNU_Trondheim/Acknowledgements/Sponsors">Acknowledgements</a> |
<a href="https://2014.igem.org/Team:NTNU_Trondheim/Project" class="flyout-toggle"><span> </span></a> | <a href="https://2014.igem.org/Team:NTNU_Trondheim/Project" class="flyout-toggle"><span> </span></a> | ||
<ul class="flyout"> | <ul class="flyout"> | ||
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<p> <ol> | <p> <ol> | ||
<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#8">Digest</a> of BioBricks <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a>.</li> | <li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#8">Digest</a> of BioBricks <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a>.</li> | ||
- | <li | + | <li>Gel electrophoresis verification of digest on agarose gel.</li> |
</ol> | </ol> | ||
</p> | </p> | ||
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<div> | <div> | ||
<h6>June 20th | <h6>June 20th | ||
- | <div | + | <div class∓ctype=text/javascript">/a> solutions for future lab work.a href="http://www.snapgene.com/resources/plasmid_files/fluorescent_protein_genes_and_plasmids/mCherry/">mCherry/div> |
+ | ∓ctype=text/javascript">/a> solutions for future lab work.a href="http://www.snapgene.com/resources/plasmid_files/fluorescent_protein_genes_and_plasmids/mCherry/">mCherry/div> | ||
+ | ="nb-onetech-i nohilite">show technical details</div> | ||
</h6> | </h6> | ||
- | <div class="nb-tech">The upstream part (J) was cut with <i>Eco</i>RI-HF and <i> | + | <div class="nb-tech">The upstream part (J) was cut with <i>Eco</i>RI-HF and <i>S∓ctype=text/javascript">/a> solutions for future lab work.pe</i>I restriction enzymes, the downstream part (B) was cut with <i>Xba</i>I and <i>Pst</i>I-HF restriction enzymes, while the psB1C3 backbone was cut with <i>Eco</i>RI-HF, <i>Pst</i>I-HF and <i>Dpn</i>I restriction enzymes. The restriction mixtures were left at 37 °C, heat killed at 80 °C for 30 minutes, then stored at -20 °C.</div> |
<p> <ol> | <p> <ol> | ||
<li>Made <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#1">LB plates</a> with chloramphenicol.</li> | <li>Made <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#1">LB plates</a> with chloramphenicol.</li> | ||
- | <li>Digest of <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts. | + | <li>Digest of <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:pSB1C3">psB1C3</a> according to the <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#11">3A assembly</a> method.</li> |
</ol> | </ol> | ||
</p> | </p> | ||
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<li>PCR amplification of <a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a> for Gibson assembly using designed primers.</li> | <li>PCR amplification of <a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a> for Gibson assembly using designed primers.</li> | ||
<li>Made two batches of <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#1">LB plates</a> with kanamycin + chloramphenicol + IPTG.</li> | <li>Made two batches of <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#1">LB plates</a> with kanamycin + chloramphenicol + IPTG.</li> | ||
- | <li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#15">Gel electrophoresis verification</a> the DNA fragments from July 16th and July 17th | + | <li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#15">Gel electrophoresis verification</a> of the DNA fragments from July 16th and July 17th. The gel still did not show expected bands.</li> |
</ol> | </ol> | ||
</p> | </p> | ||
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<p> <ol> | <p> <ol> | ||
<li>Gel verification repeated for psB1C3 backbone. DNA fragment did not appear on gel.</li> | <li>Gel verification repeated for psB1C3 backbone. DNA fragment did not appear on gel.</li> | ||
- | <li>PCR amplification of <a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a>, <a href="http://parts.igem.org/Part:pSB1K3">psB1K3 backbone</a> | + | <li>PCR amplification of (1) <a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a>, <a href="http://parts.igem.org/Part:pSB1K3">psB1K3 backbone</a>, <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#18">Kanamycin_resistance</a> previously used for Gibson assembly and (2) <a href="http://parts.igem.org/Part:pSB1K3">psB1K3 backbone</a> from the iGEM kit.</li> |
<li>PCR products purified with PCR purification kit. Concentration measured with nanodrop.</li> | <li>PCR products purified with PCR purification kit. Concentration measured with nanodrop.</li> | ||
<li>PCR products verified with gel electrophoresis.</li> | <li>PCR products verified with gel electrophoresis.</li> | ||
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</div> | </div> | ||
</div> | </div> | ||
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<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
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<li>Colonies with <a href="http://parts.igem.org/Part:BBa_J23115">BBa_J23115</a> was inoculated in 6 ml SOC ol> | <li>Colonies with <a href="http://parts.igem.org/Part:BBa_J23115">BBa_J23115</a> was inoculated in 6 ml SOC ol> | ||
with chloramphenicol.</li> | with chloramphenicol.</li> | ||
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</p> | </p> | ||
</div> | </div> | ||
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/a> | /a> | ||
/a>. | /a>. | ||
+ | /div> | ||
+ | ol> | ||
+ | /div> | ||
+ | i> Synechocystis /div> | ||
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+ | /div> | ||
+ | /li> | ||
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+ | /a> | ||
+ | ol> | ||
+ | div class="entry nb-wet"> | ||
+ | /div> | ||
+ | p> | ||
+ | li>PCR products verified with gel electrophoresis./a> of h5 class="centered">(22/09 - 28/09) | ||
+ | div class="entry nb-wet"> | ||
+ | li>PCR products purified with PCR purification kit. Concentration measured with nanodrop.h6>August 27th | ||
+ | /div> | ||
+ | |||
+ | |||
+ | p> |
Latest revision as of 21:49, 17 October 2014
Team:Cornell/notebook
From 2013.igem.org
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Week 18
(28/04 - 04/05)
April 29th
show technical details
-
Open Day at NTNU. The NTNU iGEM team explained about synthetic biology and iGEM, as well as showed and explained about some simple experimental methods (e.g. gel electrophoresis).
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
/a> solutions for future lab work.a href="http://www.snapgene.com/resources/plasmid_files/fluorescent_protein_genes_and_plasmids/mCherry/">mCherry/div>
∓ctype=text/javascript">/a> solutions for future lab work.a href="http://www.snapgene.com/resources/plasmid_files/fluorescent_protein_genes_and_plasmids/mCherry/">mCherry/div>
="nb-onetech-i nohilite">show technical details
- Made LB plates with chloramphenicol.
- Digest of BBa_J23101, BBa_B0034 and psB1C3 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
show technical details
- 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
show technical details
- 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.
July 4th
show technical details
- Ligated BBa_J23101, BBa_B0034 and psB1C3 backbone, then heat-shock transformed plasmid into competent E. coli DH5α.
- Amplified Kanamycin_resistance and Righ_flank using touchdown PCR.
July 5th
show technical details
Picked and inoculated one colony from BBa_J23101, BBa_B0034 and psB1C3 backbone ligation from July 4th.
July 6th
show technical details
- Ligated Kanamycin_resistance, Righ_flank and psB1A3 backbone, then heat-shock transformed plasmid into competent E. coli DH5α.
- Picked and inoculated three colonies from BBa_J23101, BBa_B0034 and psB1C3 backbone ligation from July 4th.
Week 28
(07/07 - 13/07)
July 7th
show technical details
- Amplification of psB1A3 backbone, psB1C3 backbone and psB1K3 backbone using touchdown PCR.
- Digestion, ligation and heat-shock transformed of Left_flank, JB with psB1A3 backbone and Kanamycin_resistance, Righ_flank with psB1A3 backbone.
July 8th
show technical details
- Made LB plates with ampicillin and with chloramphenicol.
- Heat-shock transformed synthesised promoter + RBS region (Construct) into competent E. coli DH5α.
- One colony from each ligation on July 7th were picked and inoculated.
July 9th
show technical details
- Mini-prep of inoculated colonies of Left_flank+JB and Kanamycin_resistance+Righ_flank.
- Digestion, ligation and heat-shock transformation of LFJB, BBa_C0012 and psB1C3 backbone.
- Rehydrated BioBrick BBa_E1010 and heat-shock transformed into competent E. coli DH5α.
July 10th
show technical details
- Gel electrophoresis verification of digest of Left_flank+JB, BBa_C0012, BBa_E1010 and psB1C3 backbone from July 9th on agarose gel using NotI-HF restriction enzyme.
- LFJB on psB1A3 backbone and KRF on psB1A3 backbone sent for sequencing.
- Inoculated new colonies of LFJB and KRF and BBa_E1010 because the gel verification did not give expected bands.
July 11th
show technical details
- Mini-prep of Construct and Left_flank+JB samples.
- Gel electrophoresis verification of all samples of Construct and LFJB on agarose gel using XbaI and PstI restriction enzymes.
- Picked and inoculated new colonies of BBa_E1010 and KRF.
July 12th
show technical details
- Mini-prep of BBa_E1010 and KRF solutions.
- Digest of E1010, Construct and psB1K3 backbone.
- Gel electrophoresis verification of KRF samples on agarose gel.
July 13th
show technical details
- Made new SOC medium and new LB plates with kanamycin.
- Another gel electrophoresis verification attempt with the digests from July 12th.
Week 29
(14/07 - 20/07)
July 14th
show technical details
Used a ratio of 1 in the ligation of insert and backbone.
- Gel electrophoresis verification of restriction enzymes.
- Attempted a new digestion, ligation and heat-shock transformed of Kanamycin_resistance, Righ_flank and psB1A3 backbone.
July 15th
show technical details
BBa_K576005 was found in plate 1, well 10A. BBa_K576005 equals J and B BioBrick ligated together. We decided to use this BioBrick instead of J and B separately because of all the trouble we were having.
- Gel electrophoresis verification of Left_flank, Righ_flank and Kanamycin_resistance using touchdown PCR.
- Digested RF, Kan and psB1A3 backbone.
- Rehydrated BioBrick BBa_K576005 and heat-shock transformed into competent E. coli DH5α.
July 16th
show technical details
- PCR amplification of Left_flank, BBa_C0012, BBa_E1010, Kanamycin_resistance, Righ_flank, psB1C3 backbone, BBa_K576005 and Construct for Gibson assembly using designed primers.
- Made a new batch of competent E. coli DH5α cells.
July 17th
show technical details
- PCR amplification of BBa_C0012 for Gibson assembly using designed primers.
- Made two batches of LB plates with kanamycin + chloramphenicol + IPTG.
- Gel electrophoresis verification of the DNA fragments from July 16th and July 17th. The gel still did not show expected bands.
July 18th
show technical details
- Gel verification repeated for psB1C3 backbone. DNA fragment did not appear on gel.
- PCR amplification of (1) BBa_C0012, psB1K3 backbone, Kanamycin_resistance previously used for Gibson assembly and (2) psB1K3 backbone from the iGEM kit.
- PCR products purified with PCR purification kit. Concentration measured with nanodrop.
- PCR products verified with gel electrophoresis.
- Verification of newly made competent cells were done by transforming with BBa_J23101.
July 20th
show technical details
- Colonies with BBa_C0012 was inocluated in 6 ml SOC with ampicillin.
- Colonies with BBa_J23115 was inoculated in 6 ml SOC ol> with chloramphenicol.
Week 30
(21/07 - 27/07)
July 21st
show technical details
- Mini-prep of BBa_C0012 and BBa_J23115. Concentration measured at 52.7 ng/µl for BBa_C0012 and 104 ng/µl for BBa_J23115 with nanodrop .
- PCR amplification was done with Q5-polymerase of samples consisting of PI with backbone primers, chloramphenicol backbone from mini-prep of BBa_J23115, earlier Mini-prepBBa_C0012 and original linearized plasmid with backbone primers.
- PCR products purified with PCR purification kit. Products run on gel and concentration measured with nanodrop.
July 22nd
show technical details
- heat-shock transformation of biobricks BBa_C0012 well 2N plate 2 and BBa_ I14044 well 16D plate 2 taken from kit as alternative to LacI repressor BBa_C0012.
July 23rd
show technical details
- Colony PCR of biobricks BBa_C0012 and BBa_ I14044 with Q5-polymerase.
- PCR products purified with PCR purification kit. Attempted verification with gel electrophoresis, but results were inconclusive.
July 24th
show technical details
- Colony PCR of biobricks BBa_C0012 and BBa_ I14044 with Q5-polymerase done once more, but with four different colonies.
- PCR products purified with PCR purification kit. Samples run on gel.
July 25th
show technical details
- Colony PCR of biobricks BBa_C0012, BBa_ I14044, Right Flank, Left flank, construct and chloramphenicol backbone with phusion enzyme and new primers. PCR amplification was also done with BBa_K576005 and BBa_E1010. Two duplicates were made of each sample.
- PCR products verified with gel electrophoresis. Gel showed that all parts needed for Gibson assembly were successfully made. The backbone made from BBa_C0012 was not seen on the gel.
- Duplicate products were mixed together and purified with PCR purification kit. BBa_ I14044 was discarded as BBa_C0012 showed correct bands.
- Gibson assembly was performed with the PCR products. Reaction was inoculated at 50 degrees Celsius for 60 minutes. Product stored at -20 degrees Celsius.
July 26th
show technical details
- heat-shock transformation of Gibson assembly product was done with E. coli DH5α cells.
July 27th
show technical details
- Inspection of plates from yesterday showed no growth.
Week 31
(28/07 - 03/08)
July 28th
show technical details
- Gibson assembly done once more with same products as in previous entry. Performed with 0.2 pmol of chloramphenicol backbone, and with 0.6 pmol of remaining DNA fragemnts. Gibson assembly done with a time series of 15 minutes, 30 minutes and 60 minutes.
- Partial ligation of Left Flank +BBa_ K576005, BBa_C0012 + construct + BBa_E1010 and Kanamycin_resistance + Right Flank. Verified with gel electrophoresis.
- 1 µl of Gibson product stored at -20 degrees Celsius as backup. The rest was transformed with heat-shock transformation into competent E. coli DH5α cells. Plated out on plates with kanamycin and IPTG, and on plates with chloramphenicol, kanamycin and IPTG.
July 29th
show technical details
- Plates showed one colony. Plates further incubated at 37 degrees Celsius.
- Separated the partial ligation products from yesterday with gel electrophoresis. Cut out fragments from gel and purified with gel purification kit. Concentration of fragments measured with nanodrop. Gibson assembly performed with partial ligation fragments at 50 degrees Celsius for 15 minutes.
- PCR amplification of (a) Gibson assembly done with partial ligation, (b) Gibson time series (15, 30 and 60 minutes), (c) Gibson assembly done July 25th and for backbones containing either kanamycin or chloramphenicol.
- New plates with kanamycin and IPTG and plates with kanamycin, chloramphenicol and IPTG was made.
- Transformed a biobrick from well 6B plate 4 containing kanamycin backbone with heat-shock transformation into competent E. coli DH5α cells.
July 30th
show technical details
- Gel electrophoresis done on PCR products from July 29th. Gel showed that Gibson assembly done July 25th did not result in correct assembly. The Gibson time series showed double bands on gel. The products were left in freezer. The Gibson assembly done with the partial ligation fragments were purified with PCR purification kit and concentration was measured with nanodrop (70.8 ng/µl). Chloramphenicol and kanamycin backbone verified, gel showed weaker bands for the chloramphenicol backbones.
- Gibson assembly performed with the Gibson partial ligation and chloramphenicol backbone with a reaction time of 15 minutes at 50 degrees Celsius.
- Transformed Gibson assembly product with heat-shock transformation into competent E. coli DH5α cells, except for 1 µl stored as backup.
- The single colony from the 15 minutes Gibson assembly done July 28th plated out on plate with kanamycin, chloramphenicol and IPTG and also inoculated in liquid medium overnight.
- The PCR product of the biobrick from well 6B plate 4 containing kanamycin backbone was run with gel electrophoresis. Gel showed no DNA fragments of the correct length. Inoculated in liquid medium with kanamycin overnight.
July 31st
show technical details
- Mini-prep of the inoculated colony of the Gibson 15 minutes assembly. Digested, run on gel and PCR amplified. PCR product run on gel.
Week 32
(04/08 - 10/08)
August 4th
show technical details
- Inoculation of Gibson product assembled with partial ligation in 5 ml SOC with kanamycin, chloramphenicol and IPTG. Incubated at 37 degrees Celsius overnight.
- The Gibson partial ligation was also plated out on plates containing kanamycin, chloramphenicol and IPTG.
August 5th
show technical details
- Mini-prep of inoculation from yesterday with the Gibson partial ligation. Concentration measured at 67.6 ng/µl with nanodrop.
- OD730 of Synechocystis culture was determined to be 0.346. Culture was concentrated to OD730: 2.5 before transforming with the 15 minutes Gibson PCR product.
August 6th
show technical details
- The Gibson partial ligation product was amplified with PCR with phusion enzyme.
- The amplified Gibson partial ligation product was purified with PCR purification kit.
- The purified Gibson partial ligation product was analyzed with gel electrophoresis.
Week 35
(25/08 - 31/08)
August 26th
show technical details
-
Faculty blog – Wrote a blog about iGEM for the Natural Sciences faculty at NTNU.
August 27th
show technical details
-
Interview on local radio (NRK P2 Echo) – discussing importance of molecular biology and iGEM to the global scientific community.
August 27th
show technical details
-
Assoc. Prof. Drew Endy presentation – Presented our project to students and staff of the Biotechnology Institute as a support presentation for Assoc. Prof. Drew Endy.
Week 37
(08/09 - 14/09)
September 12th
show technical details
-
PCR amplification of LF (left flank), RF (right flank) and kan (kanamycin) from Synechocystis with kanamycin resistance and pSB1C3 from biobrick E1010.
Week 38
(15/09 - 21/09)
September 16th
show technical details
-
Verification of LF, RF, Kan and pSB1C3 was done with gel electrophoresis. Samples were purified with PCR purification kit. LF, RF, Kan, pSB1C3, GOx (glucose oxidase), Lac (lac operator) was digested with EcoRI-HF and PstI-HF for 90 minutes at 37 degrees Celsius. Digests purified with PCR purification kit and stored in freezer.
September 17th
show technical details
-
Concentration of LF, RF, Kan, pSB1C3, GOx and Lac was measured with nanodrop, and amplified with PCR. Two parallels of LF, RF and Kan (no. 1 and no.2) were run. PCR products were verified with gel electrophoresis and purified with PCR purification kit. Purified products stored in freezer.
September 18th
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Digestion of LF, RF, Kan, pSB1C3, GOx, Syn: LF1, RF1, Kan, pSB1C3, GOx, Syn with EcoRI and PstI, LF2 with EcoRI and SpeI, RF2 and Kan with XbaI and PstI. Samples inoculated at 37 degrees Celsius for 90 minutes, and thereafter purified with PCR purification kit. Concentrations were measured with nanodrop. The following digests were ligated with T4-ligase: pSB1C3 + LF1 (1:3), pSB1C3 + RF1 (1:3), pSB1C3 + Kan1 (1:3), pSB1C3 + GOx (1:3), pSB1C3 + Syn (1:5), pSB1C3 + LF2 + Kan2 (1:3:3). Ligation samples left in freezer overnight.
September 19th
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Transformed yesterday’s ligations into E. coli DH5α cells. Inoculated overnight at 37 degrees Celsius.
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)
September 25th
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Four colonies from two of yesterdays plates were selected, and inoculated in 4 ml SOC with kanamycin. The same colonies were also used for colony PCR. PCR-products verified with gel electrophoresis.
September 26th
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Researcher's Night at NTNU. The NTNU iGEM team explained about synthetic biology and iGEM, as well as showed and explained about some simple experimental methods (e.g. gel electrophoresis).
September 26nd
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Mini-prep of 2 parallels of LF+Kan+RF+pSB2C3 (ligation). Digested with EcoRI and NotI. Samples run on gel, one sample verified and selected for Biobrick shipping.
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.