Miniprep for pGFPrrnB with yneA

Aims

The aim of this experiment is to produce more stocks of yneA in pGFPrrnB.

Materials and Protocol

Please refer to qiagen minipreps and nanodrop spectrophotometer protocols.

Results

The results from the nanodrop:

Discussion

The results from the nanodrop showed that we have produced high concentration of vector pGFPrrnB with insert yneA. We will then proceed to digestion.

Single and Double Digestion of pGFPrrnB with yneA

Aims

To check if the insert yneA has been inserted into vector pGFPrrnB.

Materials and Protocol

We are doing two digests for pGFPrrnB and yneA:

• Single digest with HinDIII;
• Double digest with EcoR1 and Nhe1.

Please refer to restriction digests and gel electrophoresis.

Results

Gel electrophoresis results for digestion:

Discussion and Conclusion

Single digestion of pSB1C3

Aims

To linearise the vector pSB1C3. The linear vector will be used as a PCR template for Gibson cloning of the Subtilin immunity and rocF BioBricks.

Materials and Protocol

We are doing a single digest with HinDIII.

Please refer to restriction digests and gel electrophoresis.

Results

Gel electrophoresis results for digestion:

Figure 1: Gel electrophoresis of the amplified PCR products and restriction digest

• Lane 1: 1 Kb ladder
• Lane 2: digested (linearised) pSB1C3

Discussion and Conclusion

The digestion was successful — the correct band was seen. We can proceed to gel extraction.