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Team:Warsaw/Calendar-Main/9 July 2009
From 2009.igem.org
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Isolation of BioBricks from 2008 and 2009 Kit Plates
Monika
Tasks:
- Isolate plasmids containing the biobricks
- GFP coding device switched on by IPTG - BBa_I763004 from 2008 Kit Plate 1017 well G5
- promoter lambda (cI regulated) with RFP reporter BBa_I763007 from 2009 Kit Plate 1 well 15J
- GFP generator - BBa_E0840 from 2009 Kit Plate 1 well 12O
- GFP generator - BBa_J07037 from 2009 Kit Plate 1 well 12O
Methods:
- 2009 Kit: resuspension of DNA from selected wells with 15ul of H2O
- 2008 Kit: isolation of DNA from selected wells (two punched paper spots) with 8ul of TE (prodedure described
here)
- Transformation of chemocompetent cells (prepared by Franek and Ania) with 3ul of DNA solution
- Planting on LB-agar medium supplemented with apropriate antibiotic
Results
- Will be determined tomorrow.
Creating devices to test promoters in E. coli strains (devices BBa_K177024 and BBa_K177025)
Franek
Tasks:
- Alkaline lysis of the plasmid containing BBa_I0500
- Transform competent cells with BBa_B0024
- Second attempt to transform competent cells with BBa_I0500
Methods:
- Plates with BBa_I0500 were empty, therefore once again transformation of chemocompetent cells was performed, but this time 8µl of BBa_I0500 DNA solution was used
- Plating bacterias with BBa_I0500 on LB medium supplemented with kanamycin
- Resuspension of DNA from plate 1, 2C (BBa_B0024) with 15µl of H2O
- Transformation of chemocompetent cells with 4µl of BBa_B0024 DNA solution
- Plating bacterias with 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 (BBa_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 of p53 coding sequence
Marcin
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 template
Task:
- Prepare PCR reaction to amplified p53 coding sequence.
Methods:
- PCR mixture composition:
1. 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 template, 16.5 ul MQ water
2. Negative control: the same as previously described proper mixture, the only distinction is lack of the DNA template.
- 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.
PCR products loaded into gelUsed DNA Ladder: DNA Ruler (Fermentas), 5 ul
Comment:
The effectiveness of PCR reaction was surprisingly low, probably due to degradation of the DNA template. 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
Task 1:
- Purification of amplified p53 via gel-out procedure
Methods:
- Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described here
Task 2:
- Prepare another PCR reaction to reamplified p53 coding sequence using the product of previous PCR as a template .
Methods:
- PCR mixture composition:
1. 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 template 15.5 ul MQ water
2. Negative control: the same as previously described proper mixture, the only distinction is lack of the DNA template.
- 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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