Difference between revisions of "Template:Groningen/Labjournal/Key-in-pDR111"

PCR

Experiment:

31/08/16: Amplification of the key sequence from the gBlock from IDT (see gBlocks protocol) with the primers F key only amplify and R key only amplify (see primer list) was done. The correct size of the key PCR product of 144 bp was checked with DNA electrophoresis.

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.

Conclusion:

PCR of the key sequence from the IDT gBlock was successful. We could see the correct band size which was 144 bp.

Procedure after gel validation:

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

Restriction digestion

Experiment:

31/08/16: On this day restriction digestion of the PCR product of the key sequence and pDR111 integration plasmid was performed. The key sequence as an insert was cut with SalI and HindIII restriction enzymes. The integration plasmid pDR111 as a vector was cut with exactly same restriction enzymes.

RD mixture:

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

DNA Electrophoresis:

The digestion mixture of backbone pDR111 was loaded on a gel to extract the backbone. For detailed information on how to prepare and run agarose gels see following protocol.

Conclusion:

The restriction digestion was successful. We could see a band of 7,834 bp.

Procedure after gel validation:

Digested pDR111 was cut out from the gel and DNA was extracted by Gel extraction kit (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).

Ligation

Experiment:

31/08/16: The cut key was ligated to the SalI, HindIII cut pDR111.

Ligation mixture:

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

Transformation

Experiment:

31/08/16: 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 the correct construct. The next day colonies were picked to perform colony PCR to find the correct construct with the primers F-amyE and R-amyE. Find primers here.

PCR mixture:

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

PCR set-up:

DNA Electrophoresis:

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

Conclusion:

Transformation of pDR111+key into E. coli Top10 appeared to be successful. The samples 5, 6, 7 and 9 showed the right size of band in the colony PCR. These samples were used to obtain the plasmid from an overnight culture.

Validation

Experiment:

02/09/16: Grown cultures of E. coli Top10 with pDR111+key were used to obtain a glycerol stocks and plasmid isolation was performed (QIAprep® Spin Miniprep Kit). Firstly, concentration of the plasmids obtained was measured on Nanodrop. Secondly, plasmids were sent for sequencing and then stored at -20°C.

Sequencing:

Plasmids pDR111+key from colonies 5, 6, 7 and 9 were sent for sequencing with the sequencing primer F message sequencing (see primer list).

Conclusion:

Sequencing results showed that cloning of message sequence into the integration plasmid pDR111 was successful. Sample 5, 6 and 9 show a 100% homology, so the key was successfully cloned into the pDR111 plasmid. See sequencing results in Figure 5.

Experiments

Experiments:

See Proof of concept experiment.