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Team:Warsaw/Notebook
From 2014.igem.org
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Week 1<br> | Week 1<br> | ||
| - | May 5th - May 10th, 2014 </h3 | + | May 5th - May 10th, 2014 </h3> |
| - | Let's get the show on the road!<br> | + | Let's get the show on the road!<br><br> |
Starting on a high note, we first transformed the MH1E.coli with pBAD26-PmrA/PmrB, | Starting on a high note, we first transformed the MH1E.coli with pBAD26-PmrA/PmrB, | ||
pBAD26-PmrA/PmrB-MUT, pBAD26-PmrC-GFP and pBAD26/CheZ plasmids and selected for mutants resistant to ampicillin. We also streaked the selected colonies. We followed up with transformations using the pDriveKan(m12), pDriveKan(m6), and ΔFos/pSB1C plasmids. | pBAD26-PmrA/PmrB-MUT, pBAD26-PmrC-GFP and pBAD26/CheZ plasmids and selected for mutants resistant to ampicillin. We also streaked the selected colonies. We followed up with transformations using the pDriveKan(m12), pDriveKan(m6), and ΔFos/pSB1C plasmids. | ||
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<h3><p align="justify"> | <h3><p align="justify"> | ||
Week 2 <br> | Week 2 <br> | ||
| - | May 12th - May 17th, 2014 </h3 | + | May 12th - May 17th, 2014 </h3> |
We started off by digesting the freshly procured pSB1C/ΔFos plasmid with pairs of all four BioBrick-compatible endonucleases: EcoRI, PstI, SpeI, Xbal and subsequently put them on electrophoresis to screen for possible mutations. Afterwards, pDriveKan/PmrC–GFP and pJBAD26/CheZ plasmid-carrying bacteria were inoculated for an overnighter, which we later miniprepped for future uses. Towards the end of the week, we transformed bacteria with ligations: m12, m6 and repeated streaking of E.coli/ pDriveKan(m6) and pDriveKan(m12). </p> | We started off by digesting the freshly procured pSB1C/ΔFos plasmid with pairs of all four BioBrick-compatible endonucleases: EcoRI, PstI, SpeI, Xbal and subsequently put them on electrophoresis to screen for possible mutations. Afterwards, pDriveKan/PmrC–GFP and pJBAD26/CheZ plasmid-carrying bacteria were inoculated for an overnighter, which we later miniprepped for future uses. Towards the end of the week, we transformed bacteria with ligations: m12, m6 and repeated streaking of E.coli/ pDriveKan(m6) and pDriveKan(m12). </p> | ||
<h3><p align="justify"> | <h3><p align="justify"> | ||
Week 3 <br> | Week 3 <br> | ||
| - | May 19th - May 24th, 2014 </h3 | + | May 19th - May 24th, 2014 </h3> |
We digested plasmids carrying the m6 and m12 inserts with EcoRI and PstI. Subsequently, we electrophoresised these with PstI, SpeI, Xbal, EcoRI. Later, we repeated the same procedures for the pSB1C-backboned plasmids carrying the BBa_0030 BioBrick part with both EcoRI/PstI and Xbal/SpeI. Screening for mutations returned none. </p> | We digested plasmids carrying the m6 and m12 inserts with EcoRI and PstI. Subsequently, we electrophoresised these with PstI, SpeI, Xbal, EcoRI. Later, we repeated the same procedures for the pSB1C-backboned plasmids carrying the BBa_0030 BioBrick part with both EcoRI/PstI and Xbal/SpeI. Screening for mutations returned none. </p> | ||
<h3><p align="justify"> | <h3><p align="justify"> | ||
Week 4 <br> | Week 4 <br> | ||
| - | May 26th - May 30th, 2014 </h3 | + | May 26th - May 30th, 2014 </h3> |
We transformed our bacteria E.coli MH1 with plasmids pSB1C carrying BBa_J04450 part and electrophoresized linear plasmids from the 2013 Distribution – pSB1C, pSB1A3, pS2K3 to replenish our stocks. </p> | We transformed our bacteria E.coli MH1 with plasmids pSB1C carrying BBa_J04450 part and electrophoresized linear plasmids from the 2013 Distribution – pSB1C, pSB1A3, pS2K3 to replenish our stocks. </p> | ||
<h3><p align="justify"> | <h3><p align="justify"> | ||
Week 5 <br> | Week 5 <br> | ||
| - | June 5th - June 8th, 2014 </h3 | + | June 5th - June 8th, 2014 </h3> |
At Polish universities, June usually means exams so we had little time for labwork, yet still: | At Polish universities, June usually means exams so we had little time for labwork, yet still: | ||
We transformed our dear E.coli with ligations we produced during the last week of work in May. Then, we phosphorylated linear pSB1A3, pSB1C3, pSB1T3 and pSB1K3 vectors, ligated them with PmrA/PmrB-MUT as well as PmrC-GFP and transformed these vectors into E.coli. Next we transformed bacteria ligated with plasmids pSB1C3. Then we inoculated the night bacteria with pSB1C3 carrying both PmrA/PmrB-MUT and PmrC-GFP, isolated and digested these vectors with EcoRI/PstI, and electrophoresized them for screening. Screening came out positive so it's all good... for now. </p> | We transformed our dear E.coli with ligations we produced during the last week of work in May. Then, we phosphorylated linear pSB1A3, pSB1C3, pSB1T3 and pSB1K3 vectors, ligated them with PmrA/PmrB-MUT as well as PmrC-GFP and transformed these vectors into E.coli. Next we transformed bacteria ligated with plasmids pSB1C3. Then we inoculated the night bacteria with pSB1C3 carrying both PmrA/PmrB-MUT and PmrC-GFP, isolated and digested these vectors with EcoRI/PstI, and electrophoresized them for screening. Screening came out positive so it's all good... for now. </p> | ||
Revision as of 20:26, 17 October 2014
Lab Notebook
Week 1
May 5th - May 10th, 2014
Let's get the show on the road!
Starting on a high note, we first transformed the MH1E.coli with pBAD26-PmrA/PmrB,
pBAD26-PmrA/PmrB-MUT, pBAD26-PmrC-GFP and pBAD26/CheZ plasmids and selected for mutants resistant to ampicillin. We also streaked the selected colonies. We followed up with transformations using the pDriveKan(m12), pDriveKan(m6), and ΔFos/pSB1C plasmids.
Unfortunately, the latter transformations failed which compelled us to repeat the procedure. Inoculated the ΔFos/pSB1C plasmid-carrying bacteria with fresh LB, all done with thought of procuring a nice supply of the pSB1C plasmid backbone, which we indeed succeeded in miniprepping at the end of the week.
Week 2
May 12th - May 17th, 2014
We started off by digesting the freshly procured pSB1C/ΔFos plasmid with pairs of all four BioBrick-compatible endonucleases: EcoRI, PstI, SpeI, Xbal and subsequently put them on electrophoresis to screen for possible mutations. Afterwards, pDriveKan/PmrC–GFP and pJBAD26/CheZ plasmid-carrying bacteria were inoculated for an overnighter, which we later miniprepped for future uses. Towards the end of the week, we transformed bacteria with ligations: m12, m6 and repeated streaking of E.coli/ pDriveKan(m6) and pDriveKan(m12).
Week 3
May 19th - May 24th, 2014
We digested plasmids carrying the m6 and m12 inserts with EcoRI and PstI. Subsequently, we electrophoresised these with PstI, SpeI, Xbal, EcoRI. Later, we repeated the same procedures for the pSB1C-backboned plasmids carrying the BBa_0030 BioBrick part with both EcoRI/PstI and Xbal/SpeI. Screening for mutations returned none.
Week 4
May 26th - May 30th, 2014
We transformed our bacteria E.coli MH1 with plasmids pSB1C carrying BBa_J04450 part and electrophoresized linear plasmids from the 2013 Distribution – pSB1C, pSB1A3, pS2K3 to replenish our stocks.
Week 5
June 5th - June 8th, 2014
At Polish universities, June usually means exams so we had little time for labwork, yet still:
We transformed our dear E.coli with ligations we produced during the last week of work in May. Then, we phosphorylated linear pSB1A3, pSB1C3, pSB1T3 and pSB1K3 vectors, ligated them with PmrA/PmrB-MUT as well as PmrC-GFP and transformed these vectors into E.coli. Next we transformed bacteria ligated with plasmids pSB1C3. Then we inoculated the night bacteria with pSB1C3 carrying both PmrA/PmrB-MUT and PmrC-GFP, isolated and digested these vectors with EcoRI/PstI, and electrophoresized them for screening. Screening came out positive so it's all good... for now.
