Team:NTNU Trondheim/Notebook
From 2014.igem.org
Line 674: | Line 674: | ||
<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.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:pSB1C3">psB1C3 backbone</a> according to the <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#11">3A assembly</a> method.</li> | + | li id="pt-login">div id="week23entry" class="nb-week" style="display: block"> |
+ | <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 backbone</a> according to the <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#11">3A assembly</a> method.</li> | ||
</ol> | </ol> | ||
</p> | </p> | ||
Line 1,052: | Line 1,053: | ||
<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
- | <h6> | + | <h6>July 17th |
<div class="nb-onetech-i nohilite">show technical details</div> | <div class="nb-onetech-i nohilite">show technical details</div> | ||
</h6> | </h6> | ||
Line 1,103: | Line 1,104: | ||
<p> <ol> | <p> <ol> | ||
<li>Mini-prep of inoculation from yesterday with the Gibson partial ligation. Concentration measured at 67.6 ng/µl with nanodrop.</li> | <li>Mini-prep of inoculation from yesterday with the Gibson partial ligation. Concentration measured at 67.6 ng/µl with nanodrop.</li> | ||
- | <li>OD730 of <i> Synechocystis </i> culture was determined to be 0.346. Culture was concentrated to OD730: 2.5 before transforming with the 15 minutes Gibson PCR product. </li> | + | <li>OD730 of <i> Synechocystis </i> culture was determined to be 0.346. Culture was concentrated to OD730: 2.5 before transforming with the 15 minutes Gibson PCR product. </li> /i>I-HF due to heat resistance issues with the high fidelity enzyme, the buffer used during digest had to be changed. Instead of using CutSmart buffer, Neb buffer was used for |
</ol> | </ol> | ||
</p> | </p> | ||
Line 1,418: | Line 1,419: | ||
a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#7">Mini-prep-- | a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#7">Mini-prep-- | ||
p> | p> | ||
+ | /p> |
Revision as of 12:54, 17 October 2014
Team:Cornell/notebook
From 2013.igem.org
Notebook
Filter by subteam:
show all subteams
show technical details
![](https://static.igem.org/mediawiki/2013/5/50/Cornell-nb-wet-bc.png)
![](https://static.igem.org/mediawiki/2013/2/29/Cornell-nb-dry-sc.png)
![](https://static.igem.org/mediawiki/2013/4/45/Cornell-nb-hprac-sc.png)
![](https://static.igem.org/mediawiki/2013/d/d4/Cornell-nb-wiki-sc.png)
![](https://static.igem.org/mediawiki/2013/8/85/Cornell-nb-pres-sc.png)
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. li id="pt-login">div id="week23entry" class="nb-week" style="display: block">
- 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
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 the DNA fragments from July 16th and July 17th on agarose 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. /i>I-HF due to heat resistance issues with the high fidelity enzyme, the buffer used during digest had to be changed. Instead of using CutSmart buffer, Neb buffer was used for
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 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
show technical details
-
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
show technical details
-
Transformed yesterday’s ligations into E. coli DH5α cells. Inoculated overnight at 37 degrees Celsius.
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)
September 25th
show technical details
-
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 26nd
show technical details
-
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
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.