Team:LCG-UNAM-Mexico:Journals:Nando's
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=='''Nando's lab journal'''== | =='''Nando's lab journal'''== | ||
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1.C1895 | 1.C1895 | ||
2[[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] | 2[[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] | ||
- | 3.C331 | + | 3.[[Team:LCG-UNAM-Mexico/Resources/Strains|C331]] |
4.C1906 | 4.C1906 | ||
5.C520 | 5.C520 | ||
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C 1895.- small colony spots... | C 1895.- small colony spots... | ||
[[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]].- 2 or 3 colonies. let's check for morphology | [[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]].- 2 or 3 colonies. let's check for morphology | ||
- | C331 nothing yet. let's wait a bit more. | + | [[Team:LCG-UNAM-Mexico/Resources/Strains|C331]] nothing yet. let's wait a bit more. |
C1906.- really lots of colonies. maybe the antibiotic went wrong. | C1906.- really lots of colonies. maybe the antibiotic went wrong. | ||
C520.- too many colonies. check for resistance again. | C520.- too many colonies. check for resistance again. | ||
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Plans for the day.- wait for DNTPs to perform PCR. | Plans for the day.- wait for DNTPs to perform PCR. | ||
- | Extract DNA from C1895 and C331 | + | Extract DNA from C1895 and [[Team:LCG-UNAM-Mexico/Resources/Strains|C331]] |
I'm planning the obtention of lysate from P4 sid1 this time.- | I'm planning the obtention of lysate from P4 sid1 this time.- | ||
Obtain an aliquot of P4sid 1 by diluting it in LB supplemented with calcium. | Obtain an aliquot of P4sid 1 by diluting it in LB supplemented with calcium. | ||
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I'll use smaller flasks since the fernbach doesn't fit inside our incubator. | I'll use smaller flasks since the fernbach doesn't fit inside our incubator. | ||
The bacteria grew really slow. That means, about .01 A600 in 3 hours. My best bet will be to leave the culture overnight and see what happened in the morning. | The bacteria grew really slow. That means, about .01 A600 in 3 hours. My best bet will be to leave the culture overnight and see what happened in the morning. | ||
- | I consider the experiment failed, so I'll have to try with a wt bacterium. | + | I consider the experiment failed ([[Team:LCG-UNAM-Mexico:Journals:Nando's#September 19, 2009|check plaque assay on Sep. 19]]), so I'll have to try with a wt bacterium. |
Today I also ran a pcr with various controls and primers. | Today I also ran a pcr with various controls and primers. | ||
only with int P4 primers.- | only with int P4 primers.- | ||
- | CN | + | CN<br> |
- | C1906 colony 2- expected: 3 kb band | + | C1906 colony 2- expected: 3 kb band<br> |
- | C1906 colony 3-expected: 3 kb band | + | C1906 colony 3-expected: 3 kb band<br> |
- | C1906 non-resistant, kit purified from stock-expected:3 kb band | + | C1906 non-resistant, kit purified from stock-expected:3 kb band<br> |
--- | --- | ||
P4 int and P2 ogr primers.- | P4 int and P2 ogr primers.- | ||
Line 349: | Line 344: | ||
[[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] colony 2-expected:300 bp band | [[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] colony 2-expected:300 bp band | ||
[[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] colony 3-expected:300 bp band | [[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] colony 3-expected:300 bp band | ||
- | C331 -expected: 3kb and 300 bp band | + | [[Team:LCG-UNAM-Mexico/Resources/Strains|C331]] -expected: 3kb and 300 bp band |
--- | --- | ||
P2 ogr primers only | P2 ogr primers only | ||
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[[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] colony 2- expected-nothing | [[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] colony 2- expected-nothing | ||
- | results.- a strange +- 6 b band appeared in C331 which twists my mind out. It is possible that I am retrieving a product specific for the primer combination used, so the PCR isn't really valid. Hypotheses are that this newly combined pair of primers | + | results.- a strange +- 6 b band appeared in [[Team:LCG-UNAM-Mexico/Resources/Strains|C331]] which twists my mind out. It is possible that I am retrieving a product specific for the primer combination used, so the PCR isn't really valid. Hypotheses are that this newly combined pair of primers |
- | + | ||
- | + | ||
=='''September 10, 2009'''== | =='''September 10, 2009'''== | ||
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- | Conclusion.- It seems that the stock produced from the slow growing mutant is far better that the one produced from the sole stock. I will try producing the stock with the | + | Conclusion.- It seems that the [[Team:LCG-UNAM-Mexico:Journals:Nando's#September 9, 09| stock produced]] from the [[Team:LCG-UNAM-Mexico/Resources/Strains|slow growing mutant]] is far better that the one produced from the sole stock. I will try producing the stock with the wild-type strain before keeping and characterizing this mutation. |
My plan for today was stopped, since the culture for C1895 didn't grow at all. | My plan for today was stopped, since the culture for C1895 didn't grow at all. | ||
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Gel electrophoresis.- The gel with the purification fractions is considered a complete success. Although the band seen is really thin and a bit difficult to see, the size is precisely the expected one. One thing to learn about the gel is that a high voltage and more time are required. Next time, I will try running the gel for 1:30 hrs and at 80 volts. | Gel electrophoresis.- The gel with the purification fractions is considered a complete success. Although the band seen is really thin and a bit difficult to see, the size is precisely the expected one. One thing to learn about the gel is that a high voltage and more time are required. Next time, I will try running the gel for 1:30 hrs and at 80 volts. | ||
- | Given that result, I was motivated enough | + | Given that result, I was motivated enough to run a PCR for the essential and non-essential parts of P4. The essential part is a bit over 7 kb, and the non-essential part is around 3 kb. So the PCRs were performed this way: a reaction for each of the samples containing the template band, namely DNA1, which comes from centrifuge tube 1; DNA2, from centrifuge tube 2; Fraction 5 of the purification, the elution from the column, also had the band present. |
one set of reactions attempts to amplifies P4 essential, and the other one, the non essential region. therefore, I prepared stocks for six reactions (3+3 with different primers each) and a negative control with the 7kb primers. I used lots of DNTPS so my reaction won't run out of them. | one set of reactions attempts to amplifies P4 essential, and the other one, the non essential region. therefore, I prepared stocks for six reactions (3+3 with different primers each) and a negative control with the 7kb primers. I used lots of DNTPS so my reaction won't run out of them. | ||
+ | |||
+ | [[Image:Nandogel1.jpg|200px]] | ||
+ | <br> | ||
+ | lanes 5 and 6 have a really dim band around 11 kb. | ||
=='''October 8, 2009'''== | =='''October 8, 2009'''== | ||
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The result of concentrating the phage DNA from the resting elution was completely successful. The band can be seen completely at the corresponding size and the concentration is considerably high. Restrictions and PCRs will be made with this new stock. at 9 o'clock, the restriction time will have ended. | The result of concentrating the phage DNA from the resting elution was completely successful. The band can be seen completely at the corresponding size and the concentration is considerably high. Restrictions and PCRs will be made with this new stock. at 9 o'clock, the restriction time will have ended. | ||
- | [[Image: | + | =='''October 10, 2009''== |
+ | |||
+ | [[Image:Nandogel10.jpg|200px]] | ||
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+ | Lane 8 shows the EcoRI restriction of the P4 genome. | ||
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There's something really strange going on in the restriction. Even though the DNA was considerably concentrated, after restriction with EcoRI nothing can be seen, not even the template DNA. | There's something really strange going on in the restriction. Even though the DNA was considerably concentrated, after restriction with EcoRI nothing can be seen, not even the template DNA. | ||
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- | October 13, 2009 | + | =='''October 13, 2009'''== |
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+ | So yesterday I purified DNA again, this time enhancing the technique as mentioned when I started it. | ||
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+ | The results of each DNA containing tube are here.- | ||
- | + | [[Image:Nandogel13.jpg|200px]] | |
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At the left we see the steps pre-protein denaturing. between the ladders, we observe all the DNA contained in tubes from 1 to 5. tube 3 was spilled before centrifugation, so some DNA was lost. Tube five contains a real lot of DNA, so let's hope it helps see the digestion. | At the left we see the steps pre-protein denaturing. between the ladders, we observe all the DNA contained in tubes from 1 to 5. tube 3 was spilled before centrifugation, so some DNA was lost. Tube five contains a real lot of DNA, so let's hope it helps see the digestion. | ||
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=='''October 14, 2009'''== | =='''October 14, 2009'''== | ||
Digestions were planned once again for EcoRI alone. Two more were made ith NotI and boh NotI and Xba. | Digestions were planned once again for EcoRI alone. Two more were made ith NotI and boh NotI and Xba. | ||
+ | |||
the expected bands for NotI alone are 4.5 kb and 7 kb | the expected bands for NotI alone are 4.5 kb and 7 kb | ||
+ | |||
The expected bands for NotI and XbaI are: +-2.3 kb, close to 5 kb, a 1.2 kb band and the NotI 4.5 kb band. | The expected bands for NotI and XbaI are: +-2.3 kb, close to 5 kb, a 1.2 kb band and the NotI 4.5 kb band. | ||
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- | [[Image:Nandogel14.jpg]] | + | [[Image:Nandogel14.jpg|50px]] |
- | + | The first three lanes are what matters. First observation.- the DNA sample added to the restriction was of low concentration, what c | |
1.NotI | 1.NotI | ||
bands observed.- | bands observed.- | ||
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Once with a good quantity and clues about P4 presence, there are less things to control for in the PCR. | Once with a good quantity and clues about P4 presence, there are less things to control for in the PCR. | ||
- | October 15, 2009 | + | =='''October 15, 2009'''== |
- | Today I came really early to start the protocol for chemically competent C331 and [[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] cells. The idea is use the for transformation of the cox+ogr plasmid and activate with IPTG to look for phage production as plaques. These strains have to do with P4: one assesses its production and the other one has it lisogenic along with P2. | + | Today I came really early to start the protocol for chemically competent [[Team:LCG-UNAM-Mexico/Resources/Strains|C331]] and [[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] cells. The idea is use the for transformation of the cox+ogr plasmid and activate with IPTG to look for phage production as plaques. These strains have to do with P4: one assesses its production and the other one has it lisogenic along with P2. |
- | Other thing I did today is revisit the only aparent PCR product I obtained. It was from September 9. C331 was used for a PCR containing 2 sets of primers: P4 int and ogr. The bands expected were 300 bp and 3000 bp, but the band that appeared was of 700 bp and it's quite clear. We first had discarded this as something secondary, but it didn't contradict the possibility of having a good PCR with the correct primers used. What lacked in that experiment is more combinations of DNA and primers to consistently advance with the next part. | + | Other thing I did today is revisit the only aparent PCR product I obtained. It was from September 9. [[Team:LCG-UNAM-Mexico/Resources/Strains|C331]] was used for a PCR containing 2 sets of primers: P4 int and ogr. The bands expected were 300 bp and 3000 bp, but the band that appeared was of 700 bp and it's quite clear. We first had discarded this as something secondary, but it didn't contradict the possibility of having a good PCR with the correct primers used. What lacked in that experiment is more combinations of DNA and primers to consistently advance with the next part. |
- | In response, I have planned and prepared another PCR reaction in which I prove all the primers against C331 and contrast with the results observed in the P4 sid1 genome. | + | In response, I have planned and prepared another PCR reaction in which I prove all the primers against [[Team:LCG-UNAM-Mexico/Resources/Strains|C331]] and contrast with the results observed in the P4 sid1 genome. |
Conclusion: don't fear repeating a suspicious experiment!!!! | Conclusion: don't fear repeating a suspicious experiment!!!! | ||
- | October 16.- Finally the suspition is over. I ran the PCR gel for testing P4 and P2 inside C331. Here it is.- | + | October 16.- Finally the suspition is over. I ran the PCR gel for testing P4 and P2 inside [[Team:LCG-UNAM-Mexico/Resources/Strains|C331]]. Here it is.- |
- | [[Image:Nandogel16.jpg]] | + | [[Image:Nandogel16.jpg|200px]] |
Lanes. | Lanes. | ||
1negative control (no DNA) | 1negative control (no DNA) | ||
2 cox | 2 cox | ||
3 ogr | 3 ogr | ||
- | 4 | + | 4 [[Team:LCG-UNAM-Mexico/Resources/Strains|C331]] +int |
5 ladder | 5 ladder | ||
- | 6 | + | 6 [[Team:LCG-UNAM-Mexico/Resources/Strains|C331]] + P4 |
- | 7 C331 INTp4 | + | 7 [[Team:LCG-UNAM-Mexico/Resources/Strains|C331]] INTp4 |
8 P4 DNA P4 | 8 P4 DNA P4 | ||
9 P4 DNA | 9 P4 DNA | ||
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- | October 17, 2009 | + | =='''October 17, 2009'''== |
I ran the gel testing different combinations of primers. there was no product either, so after all, we can conclude that the primers don't work. It has always been strange that both pairs of primers wouldn't work, but it seems to be the case this time. Final possibilities for lack of function.- | I ran the gel testing different combinations of primers. there was no product either, so after all, we can conclude that the primers don't work. It has always been strange that both pairs of primers wouldn't work, but it seems to be the case this time. Final possibilities for lack of function.- | ||
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- | October 19, 2009. | + | =='''October 19, 2009.'''== |
- | Yesterday I transformed C331 and [[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] cells with plasmid 18 + cox and ogr under the control of a lac operator. [[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] where the only resistants obtained, and negative controls are valid. So it's time to plan the experiment to validate the presence of cox and ogr inside the bacteria. It cannot be done from direct PCR, since [[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] already had both genes. It cannot be done only from plasmid extraction, unless the precise band is checked. the best method would be extracting plasmid and performing PCR to it. At the same time, plating on IPTG plus controls will be done, eventhough it is not sure that it will work. The expected results are P2 plaque formation. Too sad that C331 didn't transform, because we cannot observe direct interaction with P4. So growth experiment will be as follows.- | + | Yesterday I transformed [[Team:LCG-UNAM-Mexico/Resources/Strains|C331]] and [[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] cells with plasmid 18 + cox and ogr under the control of a lac operator. [[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] where the only resistants obtained, and negative controls are valid. So it's time to plan the experiment to validate the presence of cox and ogr inside the bacteria. It cannot be done from direct PCR, since [[Team:LCG-UNAM-Mexico/Resources/Strains|C2423]] already had both genes. It cannot be done only from plasmid extraction, unless the precise band is checked. the best method would be extracting plasmid and performing PCR to it. At the same time, plating on IPTG plus controls will be done, eventhough it is not sure that it will work. The expected results are P2 plaque formation. Too sad that [[Team:LCG-UNAM-Mexico/Resources/Strains|C331]] didn't transform, because we cannot observe direct interaction with P4. So growth experiment will be as follows.- |
for a plasmid containing colony, there will be these plates.- | for a plasmid containing colony, there will be these plates.- | ||
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LB with IPTG | LB with IPTG | ||
simple LB added .1 M IPTG on half of the plate. | simple LB added .1 M IPTG on half of the plate. | ||
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Latest revision as of 02:56, 21 October 2009