Isolation of BioBricks from 2008 and 2009 Kit Plates

Monika

Tasks:

• Isolate plasmids containing the biobricks

1. GFP coding device switched on by IPTG - [http://partsregistry.org/Part:BBa_I763004 BBa_I763004] from 2008 Kit Plate 1017 well G7
2. promoter lambda (cI regulated) with RFP reporter [http://partsregistry.org/Part:BBa_I763007 BBa_I763007] from 2009 Kit Plate 1 well 15J
3. GFP generator - [http://partsregistry.org/Part:BBa_E0840 BBa_E0840] from 2009 Kit Plate 1 well 12O
4. GFP generator - [http://partsregistry.org/Part:BBa_J07037 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

[http://partsregistry.org/Help:IGEM_08_DNA_distribution here])

• Transformation of chemocompetent cells (prepared by Franek and Ania) with 3ul of DNA solution
• Plating on LB medium supplemented with apropriate antibiotic

Results

• Will be determined tomorrow.

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.

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 matrix, 16.5 ul MQ water
2. 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.

Gel with the PCR product and negative control of the reaction

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

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 [http://aabiot.com/products/dna_purification/dna_fragments/gel_out/protocol_gel_out.pdf here]

Task 2:

• Prepare another PCR reaction to reamplified p53 coding sequence using the product of previous PCR as a matrix.

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 matrix, 15.5 ul MQ water
2. 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

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).