Template:Groningen/Labjournal/Key-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. 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. 50 µl PCR assay was performed according to the following
protocol. For detailed information on how to prepare and run agarose gels see
following protocol. The key sequence with prefix and suffix was successfully amplified from the gBlock. Band of 187 bp could be seen. PCR product was subsequently cleaned with (PCR Purification Kit – Jena Bioscience). 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. 20 μl RD assay was performed according to the following protocol. For detailed information on how to prepare and run
agarose gels see following protocol. 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. 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. 04/10/16: The EcoRI and PstI cut BBa_K823023 integration backbone was
ligated with the EcoRI, PstI cut key sequence. 20 µl ligation assay was performed according to the following protocol. 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. 25 µl PCR assay was performed according to the following
protocol. For detailed information on how to prepare and run agarose gel see following protocol. 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. 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.Key sequence in BBa_K823023
PCR
Experiment:
PCR mixture:
DNA Electrophoresis:
Conclusion:
Procedure after gel validation:
Restriction digestion
Experiment:
RD mixture:
DNA Electrophoresis:
Conclusion:
Procedure after gel validation:
Ligation
Experiment:
Ligation mixture:
Transformation
Experiment:
PCR mixture:
PCR set-up:
95ºC 2:00 min 95ºC 30s (30X) 60ºC 30s (30X) 72ºC 1:30 min (30X) 72ºC 2:00 min 10ºC on hold DNA electrophoresis:
Conclusion:
Validation
Experiment:
