Revision as of 12:05, 10 August 2010 by PhilipHall (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Contents 1 LacI BioBrick Construction 1.1 Aims 1.2 Materials 1.3 Protocol 1.4 Results 1.5 Conclusion

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

• Overnight digests
• Ligation mix
• Competent E. Coli DH5alpha

Protocol

• The overnight digest are run on a gel
• Repeat transformation of DH5alpha with ligation mix

Results

The gel image was unfortunately lost, however the gel was run using all of the samples and a PCR product of known length, so as to check that the Molecular weight marker was running correctly. The results were the same as the gel run yesterday, i.e. colonies 1 and 2 contain only pSB1AT3 vector (running at 3 Kbp) and the others contain pSB1AT3 with the RFP insert.

Conclusion

The conclusion that can be reached from these results is that we have not been able to get transformants with the correct plasmid/insert combination. To this end the transformation is repeated. However there is again some confusion concerning the sizes, so the samples we already have will undergo a double digest tomorrow in order to extract whatever inserts may be present.