Team:Warsaw/Calendar-Main/9 July 2009
From 2009.igem.org
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</pre> | </pre> | ||
- | After PCR the reaction was divided to 3 portion and loaded into the agarose gel to photograph and subsequently gel-out the sample | + | *Results: |
+ | After PCR the reaction was divided to 3 portion and loaded into the 1% agarose gel to photograph and subsequently gel-out the sample. | ||
+ | Used DNA Ladder: DNA Ruler (Fermentas), 5 ul | ||
[[image:PCR_p53_09_07_09.jpg |thumb|450px|center| Gel with the PCR product and negative control of the reaction]] | [[image:PCR_p53_09_07_09.jpg |thumb|450px|center| Gel with the PCR product and negative control of the reaction]] | ||
'''Comment:''' | '''Comment:''' | ||
The effectiveness of PCR reaction was surprisingly low, probably due to degradation of the DNA matrix. Reamplification of the p53 sequence using the purificated DNA sample from the gel seems to be a good idea. Of course sometimes reamplification lead to obtain partially degraded sequences but I think I should try this method. | The effectiveness of PCR reaction was surprisingly low, probably due to degradation of the DNA matrix. Reamplification of the p53 sequence using the purificated DNA sample from the gel seems to be a good idea. Of course sometimes reamplification lead to obtain partially degraded sequences but I think I should try this method. | ||
+ | |||
+ | ====Reamplification of p53==== | ||
+ | |||
+ | Tasks: | ||
+ | *Prepare another PCR reaction to reamplified p53 coding sequence using the product of previous PCR as a matrix. | ||
+ | Methods: | ||
+ | *PCR mixture composition: | ||
+ | # proper mixture: 0.25 ul primer 1 (50 nM; Oligo.pl), 0.25 ul primer 2 (50 nM; Oligo.pl), 1.5 ul dNTPs (20 uM ;Fermentas), 0.5 ul Pfu turbo polymerase (KNGiE), 2.5 ul Pfu Turbo Buffer (Fermentas), 2.5 ul MgSO<sub>4</sub> (20 uM; Fermentas), 2 ul DNA matrix, 15.5 ul MQ water | ||
+ | # Negative control: the same as previously described proper mixture, the only distinction is lack of the DNA matrix. | ||
+ | *Program: | ||
+ | p53, looked above to see details | ||
+ | *Results: | ||
+ | After PCR the reaction was divided to 2 portion and loaded into the 1% agarose gel to photograph using the UV transiluminator | ||
+ | [[image:PCR_reamplifikacja_p53_09_07_09_.png |thumb|center|Gel with the reamplified PCR ]] | ||
+ | Used DNA Ladder: DNA Ruler (Fermentas), 5 ul | ||
+ | 1,2 - PCR reaction, sample was divided to 2 portion | ||
+ | |||
+ | '''Comment:''' | ||
+ | The reaction was unsuccessful, obtained products are not specific, besides they are too short (below 500 bp). | ||
Revision as of 09:32, 12 July 2009
Contents |
Isolation of BioBricks from 2008 and 2009 Kit Plates
Monika
Selected BioBricks:
Creating devices to test promoters in E. coli strains (devices [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025])
Franek
Tasks:
- Alkaline lysis of the plasmid containing [http://partsregistry.org/Part:BBa_I0500 BBa_I0500]
- Transform competent cells with [http://partsregistry.org/Part:BBa_B0024 BBa_B0024]
- Second attempt to transform competent cells with [http://partsregistry.org/Part:BBa_I0500 BBa_I0500]
Methods:
- Plates with [http://partsregistry.org/Part:BBa_I0500 BBa_I0500] were empty, therefore once again transformation of chemocompetent cells was performed, but this time 8µl of [http://partsregistry.org/Part:BBa_I0500 BBa_I0500] DNA solution was used
- Plating bacterias with [http://partsregistry.org/Part:BBa_I0500 BBa_I0500] on LB medium supplemented with kanamycin
- Resuspension of DNA from plate 1, 2C ([http://partsregistry.org/Part:BBa_B0024 BBa_B0024]) with 15µl of H2O
- Transformation of chemocompetent cells with 4µl of [http://partsregistry.org/Part:BBa_B0024 BBa_B0024] DNA solution
- Plating bacterias with [http://partsregistry.org/Part:BBa_B0024 BBa_B0024] on LB medium supplemented with ampicillin
Results:
- Will be determined tomorrow
Jarek
Task:
- Isolation of plasmid containing parts from liquid cultures
- Digestion of acquired samples with restriction endonucleases
- Electrophoretic separation of digested samples
- Isolation of samples from agarose gel
Methods:
- Plasmid DNA was isolated with A&A "Plasmid Mini" kit, the DNA concentration was measured with nanodrop
- For digestion 1 ul of PstI/SpeI (B0032) or PstI/XbaI enzymes and 2 ul of 1xTango buffer were used. Digestion was held for 3 hours.
- After digestion samples were separated due to elecrophoresis in 0,8 agarose gel in TBE buffer
- Samples were isolated from gel with A&A "Gel-out" kit
Results:
Cloning the p53 coding sequence
Marcin
PCR of p53 coding sequence:
Comment:
Because of completely degradation of PCR product there is urgent need to amplified the p53 coding sequence. I think the original plasmid with the p53 is preserved and I'm about to repeat the PCR reaction using the plasmid as the matrix.
Tasks:
- Prepare PCR reaction to amplified p53 coding sequence.
Methods:
- PCR mixture composition:
- proper mixture: 0.25 ul primer 1 (50 nM; Oligo.pl), 0.25 ul primer 2 (50 nM; Oligo.pl), 1.5 ul dNTPs (20 uM ;Fermentas), 0.5 ul Pfu turbo polymerase (KNGiE), 2.5 ul Pfu Turbo Buffer (Fermentas), 2.5 ul MgSO4 (20 uM; Fermentas), 1 ul DNA matrix, 16.5 ul MQ water
- Negative control: the same as previously described proper mixture, the only distinction is lack of the DNA matrix.
- Program:
p53
1. 98°C - hold 2. 98°C - 2 minutes 3. 98°C - 15 sec 4. 64°C - 30 sec 5. 68°C - 2 minutes go to 2 until number of cycles=35 6. 68°C - 10 minutes 7. 4°C - hold
- Results:
After PCR the reaction was divided to 3 portion and loaded into the 1% agarose gel to photograph and subsequently gel-out the sample. Used DNA Ladder: DNA Ruler (Fermentas), 5 ul
Comment:
The effectiveness of PCR reaction was surprisingly low, probably due to degradation of the DNA matrix. Reamplification of the p53 sequence using the purificated DNA sample from the gel seems to be a good idea. Of course sometimes reamplification lead to obtain partially degraded sequences but I think I should try this method.
Reamplification of p53
Tasks:
- Prepare another PCR reaction to reamplified p53 coding sequence using the product of previous PCR as a matrix.
Methods:
- PCR mixture composition:
- proper mixture: 0.25 ul primer 1 (50 nM; Oligo.pl), 0.25 ul primer 2 (50 nM; Oligo.pl), 1.5 ul dNTPs (20 uM ;Fermentas), 0.5 ul Pfu turbo polymerase (KNGiE), 2.5 ul Pfu Turbo Buffer (Fermentas), 2.5 ul MgSO4 (20 uM; Fermentas), 2 ul DNA matrix, 15.5 ul MQ water
- Negative control: the same as previously described proper mixture, the only distinction is lack of the DNA matrix.
- Program:
p53, looked above to see details
- Results:
After PCR the reaction was divided to 2 portion and loaded into the 1% agarose gel to photograph using the UV transiluminator
Used DNA Ladder: DNA Ruler (Fermentas), 5 ul 1,2 - PCR reaction, sample was divided to 2 portion
Comment: The reaction was unsuccessful, obtained products are not specific, besides they are too short (below 500 bp).
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