Gel Electrophoresis for the Amplified Pspac_oid promoter and lacI

Aim

The aim of the experiment is to perform gel electrophoresis for the two PCR reactions which took place yesterday 5th August, 2010 and thus confirm that they were successful.

Materials and Protocol

Please refer to: Gel electrophoresis.

Result

• Lane 1: 1kb DNA ladder
• Lane 2: BioBrick compatible vector pSB1C3
• Lane 3: Pspac_oid promoter
• Lane 4: 1st fragment of rocF CDS
• Lane 5: 2nd fragment of rocF CDS
• Lane 6: 3rd fragment of rocF CDS
• Lane 7: Double Terminator
• Lane 8: 1kb DNA ladder

Table 1: Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.

Discussion

We found bands in the lanes 2,4,5,6 and 7 of the correct sizes but lane 3 did not contain any band.

Conclusion

This experiment shows that the PCR reaction was successful for all the fragments apart from Pspac_oid promoter which was present in the lane 3 and did not show any band. This could be because of the following problems:

1. Primer sequences could be incorrect.
2. Melting temperature could be incorrect.
3. Plasmid pMutin4 could have degenerated due to long term storage.

lacI ligation(repeat) and Transformations

Aims

In this experiment we aim to tranform competent E.coli DH5-alpha with pVeg spoVG and lacI in preparation for Gibson cloning. We also aim to repeat the ligation from week 12-17th July.