"
Team:NTNU Trondheim/Notebook
From 2014.igem.org
(Difference between revisions)
| Line 617: | Line 617: | ||
<div class="nb-onetech-i nohilite">show technical details</div> | <div class="nb-onetech-i nohilite">show technical details</div> | ||
</h6> | </h6> | ||
| - | <div class="nb-tech">Only 1 µl was used for the transformation, the rest of the BioBrick stock solution was stored -20 °C. BBa_J23101 was located at plate 4, 17F. BBa_B0034 was located at plate 4, 1N and BBa_C0012 was located at plate 4, 1P. Plates showed decent growth the next day; transformation a success.</div> | + | <div class="nb-tech">Only 1 µl was used for the transformation, the rest of the BioBrick stock solution was stored -20 °C. BBa_J23101 was located at plate 4, 17F. BBa_B0034 was located at plate 4, 1N and BBa_C0012 was located at plate 4, 1P. Plates showed decent growth the next day; transformation a success. The J BioBrick was meant to be used as a promotor for B and C BioBrick. B BioBrick is an RBS region and C BioBrick is the LacI repressor gene.</div> |
<p> Rehydrated BioBrick BBa_J23101, BBa_B0034 and BBa_C0012, and transformed them into competent <i>E. coli</i> DH5α cells by heat-shock, and incubated the cultures on LB plates with ampicillin overnight. | <p> Rehydrated BioBrick BBa_J23101, BBa_B0034 and BBa_C0012, and transformed them into competent <i>E. coli</i> DH5α cells by heat-shock, and incubated the cultures on LB plates with ampicillin overnight. | ||
| Line 666: | Line 666: | ||
<div class="nb-tech">The negative control showed no growth, meaning the LB plates with ampicillin + kanamycin were properly made. Plates with J, B and C BioBricks showed reasonable growth the next day.</div> | <div class="nb-tech">The negative control showed no growth, meaning the LB plates with ampicillin + kanamycin were properly made. Plates with J, B and C BioBricks showed reasonable growth the next day.</div> | ||
<p> <ol> | <p> <ol> | ||
| - | <li> | + | <li>Negative control of non-transformed <i>E. coli</i> DH5α on LB plates with ampicillin + kanamycin.</li> |
<li>Made new LB plates with ampicillin.</li> | <li>Made new LB plates with ampicillin.</li> | ||
<li>Transformed <i>E. coli</i> DH5α with BioBricks BBa_J23101, BBa_B0034 and BBa_C0012 and plated onto LB with ampicillin.</li> | <li>Transformed <i>E. coli</i> DH5α with BioBricks BBa_J23101, BBa_B0034 and BBa_C0012 and plated onto LB with ampicillin.</li> | ||
| Line 692: | Line 692: | ||
<div class="nb-onetech-i nohilite">show technical details</div> | <div class="nb-onetech-i nohilite">show technical details</div> | ||
</h6> | </h6> | ||
| - | <div class="nb-tech">J (35 bp), B (12 bp) and C (1153 bp) BioBricks all showed proper band lengths after verification on agarose gel. Note that it is not possible to physically see B and J on the gel, but the backbone attached to B and J is shown.</div> | + | <div class="nb-tech">J (35 bp), B (12 bp) and C (1153 bp) BioBricks all showed proper band lengths after verification on agarose gel. Note that it is not possible to physically see B and J on the gel, but the backbone attached to B and J is shown. The mCherry gene did not show any band on the gel, suggesting incompatibilities between the mCherry gene and the primers. Originally the mCherry gene was meant to be used as a visual confirmation of successful transformation of the final construct, but because compatibility issues with the primers we decided to explore other options. In the end we decided to use a red fluorescent protein as marker.</div> |
<p> <ol> | <p> <ol> | ||
<li>Mini-prep of PCR products from June 17th.</li> | <li>Mini-prep of PCR products from June 17th.</li> | ||
Revision as of 11:56, 12 August 2014
"
Team:Cornell/notebook
From 2013.igem.org
Notebook
Filter by subteam:
show all subteams
show technical details
"_"
Wet Lab
Dry Lab
Human Practices
Wiki
Presenters
only
only
only
only
only
Week 23
(02/06 - 08/06)
June 3rd
show technical details
- Prepared SOC and yB solutions for future lab work.
- Sterilized material and solutions needed for future lab work.
June 4th
show technical details
{{{tech}}}
Made LB plates with ampicillin and ampicillin + kanamycin.
June 5th
show technical details
{{{tech}}}
Inoculated E. coli DH5α in SOC medium overnight.
June 6th
show technical details
OD of culture was 0.3160 after 100 minutes.
Made competent E. coli DH5α cells.
Week 24
(09/06 - 15/06)
June 10th
show technical details
Transformation was done by heat-shock. A plasmid containing ampicillin resistance was used, and the transformed cells were incubated overnight on LB plates with ampicillin. Plates showed a bacterial blanket the next day; the cells were apparently super competent.
Test of transformation efficiency of competent E. coli DH5α from June 6th.
June 11th
show technical details
Only 1 µl was used for the transformation, the rest of the BioBrick stock solution was stored -20 °C. BBa_J23101 was located at plate 4, 17F. BBa_B0034 was located at plate 4, 1N and BBa_C0012 was located at plate 4, 1P. Plates showed decent growth the next day; transformation a success. The J BioBrick was meant to be used as a promotor for B and C BioBrick. B BioBrick is an RBS region and C BioBrick is the LacI repressor gene.
Rehydrated BioBrick BBa_J23101, BBa_B0034 and BBa_C0012, and transformed them into competent E. coli DH5α cells by heat-shock, and incubated the cultures on LB plates with ampicillin overnight.
June 12th
show technical details
The SOC medium was clear the next day, meaning no growth.
Inoculated BioBrick colonies from June 11th in SOC medium containing ampicillin.
June 13th
show technical details
The failed inoculation attempt on June 12th could indicate something wrong with the LB plates with ampicillin. Growth of non-transformed cells supported this. Most likely the ampicillin was aliquotted to the medium at an elevated temperature, causing denaturation of the antibiotic.
Negative control of non-transformed E. coli DH5α on LB plates with ampicillin.
Week 25
(16/06 - 22/06)
June 16th
show technical details
The negative control showed no growth, meaning the LB plates with ampicillin + kanamycin were properly made. Plates with J, B and C BioBricks showed reasonable growth the next day.
- Negative control of non-transformed E. coli DH5α on LB plates with ampicillin + kanamycin.
- Made new LB plates with ampicillin.
- Transformed E. coli DH5α with BioBricks BBa_J23101, BBa_B0034 and BBa_C0012 and plated onto LB with ampicillin.
June 17th
show technical details
These DNA sequences were to be used in the final construct. Left_flank and Right_flank sequences were needed to make conjugation of construct with the genome of Synechocystis PCC. 6803 possible. Kanamycin_resistance optimized for Synechocystis was needed in order to select for colonies with conjugated final construct. Successful PCR amplification of Left_flank (556 bp) and Kanamycin_resistance (944 bp) sequence. Right_flank (544 bp) sequence amplification failed.
- PCR amplification of Left_flank, Righ_flank and Kanamycin_resistance with Synechocystis PCC. δslr0906 as template using touchdown PCR.
- Verification of PCR products on agarose gel
June 18th
show technical details
J (35 bp), B (12 bp) and C (1153 bp) BioBricks all showed proper band lengths after verification on agarose gel. Note that it is not possible to physically see B and J on the gel, but the backbone attached to B and J is shown. The mCherry gene did not show any band on the gel, suggesting incompatibilities between the mCherry gene and the primers. Originally the mCherry gene was meant to be used as a visual confirmation of successful transformation of the final construct, but because compatibility issues with the primers we decided to explore other options. In the end we decided to use a red fluorescent protein as marker.
- Mini-prep of PCR products from June 17th.
- PCR amplification of mCherry gene, BioBricks BBa_J23101, BBa_B0034 and BBa_C0012 using touchdown PCR.
- Verification of PCR products on agarose gel
June 19th
show technical details
J and B BioBricks were digested with XbaI, while C BioBrick was digested with NotI-HF. Digest of J, B and C should have gievn a band of 2989 bp, 2097 bp and 2063 bp respectively on the gel; low concentration of DNA after digest, but all samples had correct band lengths.
- Digest of BioBricks BBa_J23101, BBa_B0034 and BBa_C0012.
- Verification of digest on agarose gel.
June 20th
show technical details
The upstream part (J) was cut with EcoRI-HF and SpeI restriction enzymes, the downstream part (B) was cut with XbaI and PstI-HF restriction enzymes, while the psB1C3 backbone was cut with EcoRI-HF, PstI-HF and DpnI restriction enzymes. The restriction mixtures were left at 37 °C for one hour, heat killed at 80 °C for 30 minutes, then stored at -20 °C.
- Made LB plates with chloramphenicol.
- Digest of BBa_J23101, BBa_B0034 and psB1C3 plasmid backbone according to the 3A Assemble method.
