Difference between revisions of "Template:Groningen/Labjournal/Key-in-BBa K823023"

 
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<h3>Key sequence in BBa_K823023</h3>
 
<h3>Key sequence in BBa_K823023</h3>
  
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<p><a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a> is an available BioBrick from igem Munich 2012. It is an  
+
<p><a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a> is an available BioBrick from iGEM Munich 2012. It is an  
 
integration plasmid for <em>Bacillus subtilis</em>, which can be used for  
 
integration plasmid for <em>Bacillus subtilis</em>, which can be used for  
 
cloning in <em>E. coli</em> as well. An RFP is inserted in BBa_K823023 for more  
 
cloning in <em>E. coli</em> as well. An RFP is inserted in BBa_K823023 for more  
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<figcaption>Figure 2. BBa_K823023 cut with EcoRI and PstI. Backbone  
 
<figcaption>Figure 2. BBa_K823023 cut with EcoRI and PstI. Backbone  
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~6000 bp, RFP insert ~1000 bp.</figcaption>
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~6,000 bp, RFP insert ~1,000 bp.</figcaption>
 
</figure>
 
</figure>
  
 
<h5> Conclusion: </h5>  
 
<h5> Conclusion: </h5>  
 
<p>The digestion of BBa_K823023 showed the correct bands on  
 
<p>The digestion of BBa_K823023 showed the correct bands on  
−
the gel for the 6000 bp backbone and the 1000 bp RFP insert.  
+
the gel for the 6,000 bp backbone and the 1,000 bp RFP insert.  
 
Therefore the cloning procedure could proceed.</p>
 
Therefore the cloning procedure could proceed.</p>
  
 
<h5>Procedure after gel validation:</h5>
 
<h5>Procedure after gel validation:</h5>
  
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<p>Digested sample of the backbone ~6000 bp were cut out from the  
+
<p>Digested sample of the backbone ~6,000 bp were cut out from the  
 
gel and DNA was extracted by Gel extraction kit (Nucleospin)
 
gel and DNA was extracted by Gel extraction kit (Nucleospin)
 
<a href="https://2016.igem.org/Team:Groningen/Protocols#Gel-extract-Nucleospin">see protocol</a>. </p>
 
<a href="https://2016.igem.org/Team:Groningen/Protocols#Gel-extract-Nucleospin">see protocol</a>. </p>

Latest revision as of 20:40, 19 October 2016

Key sequence in BBa_K823023

BBa_K823023 is an available BioBrick from iGEM Munich 2012. It is an integration plasmid for Bacillus subtilis, which can be used for cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more efficient screening after transformation. It was chosen to construct the Bacillus subtilis key strain. Construction was performed as described in the following.

PCR

Experiment:

27/09/16: The key sequence was amplified (see PCR protocol) from the pDR111+key plasmid with the primers key only + prefix and key only + suffix (primer sequences can be found here). The correct size of 187 bp of the product was checked by DNA electrophoresis. The PCR product was stored at 4°C.

PCR mixture:

50 µl PCR assay was performed according to the following protocol.

DNA Electrophoresis:

For detailed information on how to prepare and run agarose gels see following protocol.

Figure 1. DNA electrophoresis of amplified key sequence (187 bp in size).
Conclusion:

The key sequence with prefix and suffix was successfully amplified from the gBlock. Band of 187 bp could be seen.

Procedure after gel validation:

PCR product was subsequently cleaned with (PCR Purification Kit – Jena Bioscience).

Restriction digestion

Experiment:

04/10/16: The key sequence PCR product was digested with EcoRI and PstI as well as the BBa_K823023 plasmid. The digestion of the BBa_K823023 backbone was checked with DNA electrophoresis. The digestion of the key sequence PCR product was immediately cleaned with PCR Purification Kit – Jena Bioscience see protocol.

RD mixture:

20 μl RD assay was performed according to the following protocol.

DNA Electrophoresis:

For detailed information on how to prepare and run agarose gels see following protocol.

Figure 2. BBa_K823023 cut with EcoRI and PstI. Backbone ~6,000 bp, RFP insert ~1,000 bp.
Conclusion:

The digestion of BBa_K823023 showed the correct bands on the gel for the 6,000 bp backbone and the 1,000 bp RFP insert. Therefore the cloning procedure could proceed.

Procedure after gel validation:

Digested sample of the backbone ~6,000 bp were cut out from the gel and DNA was extracted by Gel extraction kit (Nucleospin) see protocol.

Ligation

Experiment:

04/10/16: The EcoRI and PstI cut BBa_K823023 integration backbone was ligated with the EcoRI, PstI cut key sequence.

Ligation mixture:

20 µl ligation assay was performed according to the following protocol.

Transformation

Experiment:

04/10/16: The ligation mix was heat shock transformed to competent Top10 E. coli cells following the protocol. Cells were plated on 100 μg/ml ampicillin LB agar to select for the correct constructs. The next day colonies were selected to perform colony PCR in order to find the correct constructs.

Figure 3. Colonies of E.coli Top10 after transformation with BBa_K823023 with the key sequence.
PCR mixture:

25 µl PCR assay was performed according to the following protocol.

PCR set-up:
95ºC2:00 min
95ºC30s(30X)
60ºC30s(30X)
72ºC1:30 min(30X)
72ºC2:00 min
10ºC on hold
DNA electrophoresis:

For detailed information on how to prepare and run agarose gel see following protocol.

Conclusion:

The transformation of the key sequence in BBa_K823023 to E. coli Top10 was successful. We have obtained correct insert size when colony PCR was done. And that was 187 bp.

Validation

Experiment:

05/10/16 Grown cultures of E. coli Top10 with the key sequence in BBa_K823023 were used for making the (see Mini prep protocol). Firstly, concentration of the plasmids obtained was measured on Nanodrop. Secondly, plasmids were sent for sequencing and then stored at -20°C.

Figure 4. Selecting colonies for glycerol stocks.

Experiments

Experiments:

See Proof of concept experiment.