Team:Dundee/CGCraigon

CSI: DUNDEE

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Nasal Mucus
Week Beginning 15/6/2015

Summary

Produce a plasmid preperation of OBP2A in the IDT plasmid

17/6: Transformation of pIDT-OBP2A in MC1610 e.coli Strain

Aim of Experiment: To transform pIDT-OBP2A into MC1061 e.coli strain

Protocols Used: Transformation

Results: N/A

Next Steps: If the transformation is successful the next step will be to produce overnights in preperation for plamsid miniprep.

18/6: Produced overnights from positive colonies of pIDT-OBP2A.

Aim of experiment: To produce overnights of pIDT-OBP2A in MC1061.

Protocols Used: Overnight Cultures

Results: N/A

Next Steps: A plasmid preperation of pIDT-OBP2A will be done next.

19/6: Plasmid Purification of pIDT-OBP2A overnight culture from yesterday.

Aim of experiment: Yesterdays overnight culture was succesful and will now undergo plasmid purification in preperation for use in PCR.

Protocols Used: Miniprep

Results: The plasmid preperation yeilded a concentration of 305.53 ng/ul.

Next Steps: A PCR using the purified pIDT-OBP2A plasmid as the template will be done tomorrow.

Week Beginning 22/6/2015

Summary

Removal of the signaling peptide from the start of OBP2A and attempt to insert OBP2A into the biobrick vector pSB1C3.

8/6: Amplification of LbpA into pQE80-L

Aim of experiment: To amplify LbpA for subsequent cloninng into the pQE80-L vector.

Protocols Used: PCR

Results: Figure 2

Next Steps: The PCR product will be digested and ligated into pQE80-L tomorrrow.

20/6: PCR of the pIDT-OBP2A plasmid using primers designed for the removal of the signalling peptide.

Aim of experiment: To set up a PCR using pIDT-OBP2A using priers that if succesful will remove the signalling peptide from the n terminus of OBP2A.

Protocols Used: PCR

Results: N/A

Next Steps: The next step will be to gel purify and extract the OBP2A PCR product from the PCR mixture. Ths will most likely be done tomorrow.

21/6: Gel extraction of OBP2A from the PCR product and a confirmatory gel image of the purified OBP2A.

Aim of experiment: To extract OBP2A from the PCR product using gel extraction. Then to subsequently image the extracted solution to determine the presence of OBP2A.

Protocols Used: Gel Extraction

Results: N/A

Next Steps: The gel image shows a band corresponding to the size of OBP2A, The next step will be to restrict this gel extracted OBP2Ain preperation for ligation.

Week Beginning 8/6/2015

Summary

Sequencing confirmed that LbpA had been successfully cloned into pSB1C3 therefore this week cloning was attempted to put LbpA into the high expression vector pQE80-L.

8/6: Amplification of LbpA into pQE80-L

Aim of experiment: To amplify LbpA for subsequent cloninng into the pQE80-L vector.

Protocols Used: PCR

Results: Figure 2

Next Steps: The PCR product will be digested and ligated into pQE80-L tomorrrow.