Team:LCG-UNAM-Mexico:Journals:Uriel
From 2009.igem.org
(Difference between revisions)
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the using this information to add the right amount of restriction enzymes. | the using this information to add the right amount of restriction enzymes. | ||
- | The first three lanes are [http://partsregistry.org/Part:BBa_K242002 cox], [http://partsregistry.org/Part:BBa_K242001 ogr] and [http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | The first three lanes are [http://partsregistry.org/Part:BBa_K242002 cox], [http://partsregistry.org/Part:BBa_K242001 ogr] and [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 amplified using the prefix and suffix |
primer for these genes | primer for these genes | ||
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This plasmid that is going to be ligated with [http://partsregistry.org/Part:BBa_K242001 ogr] is digested EcoRI/PstI. | This plasmid that is going to be ligated with [http://partsregistry.org/Part:BBa_K242001 ogr] is digested EcoRI/PstI. | ||
- | [http://partsregistry.org/Part:BBa_K242002 cox] was cut with EcoRI/SpeI and the plasmid that contain [http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | [http://partsregistry.org/Part:BBa_K242002 cox] was cut with EcoRI/SpeI and the plasmid that contain [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 with EcoRI/XbaI because we are |
- | going to insert [http://partsregistry.org/Part:BBa_K242002 cox] into this plasmid in order to concatenate [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | going to insert [http://partsregistry.org/Part:BBa_K242002 cox] into this plasmid in order to concatenate [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015. |
[[Image:14ago09-ogrEP-18EP-coxES-12EX.jpg|300px]] | [[Image:14ago09-ogrEP-18EP-coxES-12EX.jpg|300px]] | ||
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We run a 1% Agarose gel at 85 V. 1hr | We run a 1% Agarose gel at 85 V. 1hr | ||
- | Unfortunately [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | Unfortunately [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 cloning by fusing [http://partsregistry.org/Part:BBa_K242002 cox] and [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 in another plasmid |
didn't work out. We will try to repeat the procedure to see if we are luckier. | didn't work out. We will try to repeat the procedure to see if we are luckier. | ||
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- | Again we dephophorylated vector I-09#017 to ligate it with [http://partsregistry.org/Part:BBa_K242002 cox] and [http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | Again we dephophorylated vector I-09#017 to ligate it with [http://partsregistry.org/Part:BBa_K242002 cox] and [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 and then perform a transformation |
Dephosphorylation Reaction: | Dephosphorylation Reaction: | ||
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Colony PCR reactions were done with selected colonies that resulted from the last transformation | Colony PCR reactions were done with selected colonies that resulted from the last transformation | ||
the resulting and tow of the twenty-one looked like they contained [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+[http://partsregistry.org/Part:BBa_B0015 BBa_B0015] verification | the resulting and tow of the twenty-one looked like they contained [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+[http://partsregistry.org/Part:BBa_B0015 BBa_B0015] verification | ||
- | with a restriction assay was done and also [http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | with a restriction assay was done and also [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015+18 was purified and opened digested with EcoRI/PstI to be prepared |
in case last ligation don't work. | in case last ligation don't work. | ||
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== September 15, 2009 == | == September 15, 2009 == | ||
- | The ligation didn't work, I planned to follow a different strategy first purify the [http://partsregistry.org/Part:BBa_K242002 cox] fragment using Low melt point Agarose, we are using this procedure because the plasmido that contain [http://partsregistry.org/Part:BBa_K242002 cox] is the samen as the one that has [http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | The ligation didn't work, I planned to follow a different strategy first purify the [http://partsregistry.org/Part:BBa_K242002 cox] fragment using Low melt point Agarose, we are using this procedure because the plasmido that contain [http://partsregistry.org/Part:BBa_K242002 cox] is the samen as the one that has [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 and |
- | as a consequence the same antibiotic resistance gene, so by separating the DNA by electrophoresis we can take apart [http://partsregistry.org/Part:BBa_K242002 cox] gene and the ligated in to the dephoshorylated plasmid [http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | as a consequence the same antibiotic resistance gene, so by separating the DNA by electrophoresis we can take apart [http://partsregistry.org/Part:BBa_K242002 cox] gene and the ligated in to the dephoshorylated plasmid [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 that was digested EcoRI/XbaI, the resultin plasmid will be [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015. |
== September 22, 2009 == | == September 22, 2009 == | ||
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== September 23, 2009 == | == September 23, 2009 == | ||
- | A ligation is going to be done with [http://partsregistry.org/Part:BBa_K242002 cox] and [http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | A ligation is going to be done with [http://partsregistry.org/Part:BBa_K242002 cox] and [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015, [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015+18 was dephosphorylated and [http://partsregistry.org/Part:BBa_K242002 cox] was purified via gel. As a control we will try to religate the dephosphorylated plasmid, the same plasmid not dephosphorylated and a uncut plasmid |
which must transform because was not digested. | which must transform because was not digested. | ||
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T4 ligase 1µL | T4 ligase 1µL | ||
Buffer 2µL | Buffer 2µL | ||
- | [http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 2µL |
[http://partsregistry.org/Part:BBa_K242002 cox] 5µL | [http://partsregistry.org/Part:BBa_K242002 cox] 5µL | ||
Water 10µL | Water 10µL | ||
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== September 24, 2009 == | == September 24, 2009 == | ||
- | All controls resulted as expected so the must probable is that we achieved to ligate [http://partsregistry.org/Part:BBa_K242002 cox] with [http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | All controls resulted as expected so the must probable is that we achieved to ligate [http://partsregistry.org/Part:BBa_K242002 cox] with [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015+18 |
I prepared overnights to extract plasmid tomorrow and perform a restriction assay to see if we have the fragment | I prepared overnights to extract plasmid tomorrow and perform a restriction assay to see if we have the fragment | ||
of interest. | of interest. | ||
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Restriction Reactions: | Restriction Reactions: | ||
- | 1)XbaI/PstI [http://partsregistry.org/Part:BBa_K242002 cox]+18 2)XbaI/PstI [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | 1)XbaI/PstI [http://partsregistry.org/Part:BBa_K242002 cox]+18 2)XbaI/PstI [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 colonies[1-6] |
Buffer 2 2µL Buffer2 2µL | Buffer 2 2µL Buffer2 2µL | ||
BSA 0.2µL BSA 0.2µL | BSA 0.2µL BSA 0.2µL | ||
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[[Image:Gel resuelto.JPG|240px]] | [[Image:Gel resuelto.JPG|240px]] | ||
- | As the image show on the first 7 lanes we were able to ligate [http://partsregistry.org/Part:BBa_K242002 cox] with [http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | As the image show on the first 7 lanes we were able to ligate [http://partsregistry.org/Part:BBa_K242002 cox] with [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015+18 every of the |
selected clones has the [http://partsregistry.org/Part:BBa_K242002 cox] insert so this procedure was faster than the fisr that we tried to use | selected clones has the [http://partsregistry.org/Part:BBa_K242002 cox] insert so this procedure was faster than the fisr that we tried to use | ||
the only thing is that you need to purify the insert via an Agarose gel and the ligate it to the | the only thing is that you need to purify the insert via an Agarose gel and the ligate it to the | ||
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T4 ligase 1µL | T4 ligase 1µL | ||
Buffer 2µL | Buffer 2µL | ||
- | [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 5µL |
iptg 5µL | iptg 5µL | ||
Water 10µL | Water 10µL | ||
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Controls were inconsistent in particular the re-ligation of dephosphorylated plasmid which gave | Controls were inconsistent in particular the re-ligation of dephosphorylated plasmid which gave | ||
- | the same amount of colonies compared with ligation of [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | the same amount of colonies compared with ligation of [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 and 12 so we are going to |
re-dephosphorylate the vector 12 and perform a new ligation reaction. | re-dephosphorylate the vector 12 and perform a new ligation reaction. | ||
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- | The samples that we are looking on the gel correspon to [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | The samples that we are looking on the gel correspon to [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015+18 digested with XbaI/SpeI and 12 |
Once we checked concetrations we can do the ligation reaction. | Once we checked concetrations we can do the ligation reaction. | ||
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T4 ligase 1µL | T4 ligase 1µL | ||
Buffer 2µL | Buffer 2µL | ||
- | [http://partsregistry.org/Part:BBa_K242002 cox]+ | + | [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 12µL |
12 4µL | 12 4µL | ||
Water 1µL | Water 1µL | ||
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[[Image:12oct09-iptg-cox+ogr+ter-final.jpg|200px]] | [[Image:12oct09-iptg-cox+ogr+ter-final.jpg|200px]] | ||
- | The insert looks shifted upwards but a little amount so we didn't achieve to ligate the iptg to [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | The insert looks shifted upwards but a little amount so we didn't achieve to ligate the iptg to [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 |
this is strange because the plasmid maintained its size so the promoter is not there.I've checked the plasmid size an it is 2079 bp so the plasmid doesn't has the promoter. | this is strange because the plasmid maintained its size so the promoter is not there.I've checked the plasmid size an it is 2079 bp so the plasmid doesn't has the promoter. | ||
Going back further when I digested for the first time plasmid 12 I did it with SpeI/PstI to allow | Going back further when I digested for the first time plasmid 12 I did it with SpeI/PstI to allow | ||
- | promoter to be at the beginning of the construction.The insert [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | promoter to be at the beginning of the construction.The insert [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 was digested XbaI/PstI so I think the unique alternative is that promoter ipteg wasn't |
in the plasmid. | in the plasmid. | ||
- | In this circumstance we can only say that we changed [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | In this circumstance we can only say that we changed [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 from plasmid 18 to plasmid 12 that is resitantant to |
Amp. | Amp. | ||
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[http://partsregistry.org/Part:BBa_K242002 cox] | [http://partsregistry.org/Part:BBa_K242002 cox] | ||
[http://partsregistry.org/Part:BBa_K242001 ogr] | [http://partsregistry.org/Part:BBa_K242001 ogr] | ||
- | [http://partsregistry.org/Part:BBa_K242001 ogr]+[http://partsregistry.org/Part:BBa_K242002 cox]+ | + | [http://partsregistry.org/Part:BBa_K242001 ogr]+[http://partsregistry.org/Part:BBa_K242002 cox]+BBa_B0015 |
multipromotor | multipromotor | ||
Revision as of 01:09, 22 October 2009