15 July 2010

LacI BioBrick Construction

Aims

• To use PCR to extract lacI (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).

Materials

• Original transformants
• Second round of transformants
• Plasmid extracts
• Restriction enzymes Xba1 aand Pst1

Protocol

• Single (Xba1) and Double (Xba1 aand Pst1) digest of Plasmid extracts
• Run digests on a gel
• Overnight culture of all transformants

Results

Figure 1.

Discussion

This gel is unclear in precisely what is present in the samples it can clearly be seen that there are in fact two bands in the single digest lanes (where there should only be one) and three bands in the double digest lane (where there should only be two).

Conclusion

It is our current belief that the plasmid extraction may have been performed incorrectly (there was one minor mistake during the procedure), so to this end an overnight culture of the original transformant colonies, as well as second round of transformants, will be performed, ready for plasmid extraction tomorrow.