Difference between revisions of "Template:Groningen/Labjournal/Key-in-pSB1C3"

PCR of key from key in pDR111

Experiment:

27/09 The key was PCR amplified see PCR protocol Team:Groningen/Protocols#PCR-Q5 from the pDR111+key plasmid with the primers key prefix key suffix. (primer sequences can be found here) /Team:Groningen/Labjournal/PrimerList The correct size 187 bp of the product was checked by gel electrophoresis. The PCR product was stored at 4 °C.

PCR mixture:

50 μl PCR assay was performed according to the following protocol: link to PCR protocol PCR (Q5® High-Fidelity).

DNA Electrophoresis:

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

Conclusion:

The key was successfully amplified with prefix and suffix from the plasmid.

Procedure after gel validation:

PCR product was subsequently cleaned with DNA Clean-up (PCR Purification Kit – Jena Bioscience)

Restrictions digestion of key PCR product and pSB1C3+J04450

Experiment:

28/09 The key was cut with EcoRI and PstI. The backbone pSB1C3 + J04450 was digested with the same enzymes. This construct was used for easier screening after transformation because J04450 is a RFP. So red colonies could immediately be identified as once with unsuccessful digested plasmid.

RD mixture:

30 μl RD assay was performed according to the following protocol: Restriction digestion (FastDigest enzymes).

DNA Electrophoresis:

The digestion mixture of backbone pSB1C3 + J04450 was loaded on a gel to extract the backbone.

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

Conclusion:

The digestion was successful because bands for both expected fragments could be seen on the gel, namely RFP insert is 1069bp and the pSB1C3 backbone is 2019 bp.

Procedure after gel validation:

Digested pSB1C3 the upper band by 2000 bp was cut out from the gel and DNA was extracted by Gel extraction (Agarose Gel Extraction Kit – Jena Bioscience). The PCR product of the key was not checked on the gel after the digestion but immediately cleaned up with DNA Clean-up (NucleoSpin® Gel and PCR Clean-up). The concentration of the digested and cleaned key sequence was measured with the Nano drop, 10,3 ng/μl were obtained.

Ligation

Experiment:

07/10 The EcoRI, PstI cut pSB1C3 backbone was ligated with the EcoRI, PstI cut key.

Ligation mixture:

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

Transformation

Experiment:

08/10 The ligation mix was heat shock transformed to competent Top 10 E.coli cells following the protocol Transformation of E. coli (Standard protocol). Cells were plated on 50 μg/ml chloramphenicol LB agar to select the correct construct. The next day colonies were picked to perform colony PCR to find the correct construct with the primers key only prefix and key only suffix. Find primers here.

PCR mixture:

25 μl PCR assay was performed according to the following protocol: PCR (Q5® High-Fidelity).

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:

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

Conclusion:

The transformation of the key in pSB1C3 to E.coli Top 10 was successful.

Validation

Experiment:

09/10 Grown cultures of E.coli Top10 with the construct key in pSB1C3 were used to obtain Glycerol stocks Glycerol stock and plasmid isolation was performed (see QIAprep protocol) Plasmid mini-prep (Fast-n-Easy Plasmid Mini-Prep Kit - Jena Bioscience). Firstly, concentration of the plasmids obtained was measured on Nanodrop. Secondly, plasmids were sent for sequencing and then stored at -20°C.

Sequencing:

The plasmid key in pSB1C3 from colonies 1 and 3 was send for sequencing with the primers VF2 and VR, see primer list /Team:Groningen/Labjournal/PrimerList.

Conclusion:

The sequencing result proofed the successful integration of the key in the pSB1C3.

Experiments

See integration of the key in B. Subtilis via the BioBrick K823023 integration plasmid.