Team:Dundee/CGCraigon
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Summary
Produced a plasmid preperation of pIDT-OBP2A in order to use it as template in PCR.
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 an overnight culture for plasmid preparation.
Aim of experiment: To produce an overnight culture of pIDT-OBP2A from positive colonies of MC1061 E.coli strain from the transformation done on the 17/6.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: Produce a plasmid preparation of pIDT-OBP2A from this overnight culture.
Aim of experiment: The overnight culture produced yesterday will now undergo a plasmid preparation in order to obtain the pIDT-OBP2A plasmid.
Protocols Used: Miniprep
Results: The plasmid preperation yeilded a concentration of 305.53 ng/ul.
Next Steps: To run a PCR using the purified pIDT-OBP2A plasmid as a template for amplification.
Summary
Removal of the signaling peptide from OBP2A and insertion of OBP2A into the biobrick vector pSB1C3.
Aim of Experiment: To perform a PCR on the plasmid preparation of pIDT-OBP2A produced last week, using primers that have been designed in a way as to remove the signalling peptide at the start of the OBP2A gene.
Protocols Used: PCR
Results: N/A
Next Steps: The next step will be to run the PCR product on a gel and perform a gel extraction on the part of the gel corresponding to the size of the OBP2A gene fragment.
Aim of experiment: To perform a gel extraction of OBP2A from a PCR reaction mixture that was performed on the 22/6. Once gel extraction has been performed a subsequent gel will be done to determine if the gel extraction has been successful.
Protocols Used: Gel extraction
Results: As it can be seen from the gel image, a band is present corresponding to the size of the OBP2A gene fragment.
Next Steps: To perform a restriction of OBP2A in preparation for ligation.
Aim of experiment: To firstly restrict the gel extracted OBP2A. Secondly, to then ligate the restricted OBP2A into the biobrick vector pSB1C3. Third and finally to transform this plasmid into the JM110 E.coli strain.
Protocols Used: Restriction Digest : Ligation : Transformation
Results: N/A
Next Steps:If the transformation is successful and positive colonies form on the agar plate then these colonies will then be grown overnight in preparation for Miniprep.
Aim of experiment: The transformation performed on the 24/6 failed and as a result OBP2A will again ligated into the biobrick vector pSB1C3 and then transformed into JM110 E.coli strain.
Protocols Used: Ligation: Transformation
Results: N/A
Next Steps:If the transformation is successful and positive colonies form on the agar plate then these colonies will then be grown overnight in preparation for Miniprep.
Aim of experiment: To produce overnight cultures of pSB1C3-OBP2A from positive colonies of JM110 E.coli strain from the transformation done on the 25/6.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: Produce a plasmid preparation of pSB1C3-OBP2A in preparation for pre-sequence digest.
Aim of experiment: To perform a plasmid preparation of overnight cultures of pSB1C3-OBP2A from positive colonies of JM110 E.coli strain from the transformation done on the 25/6. Then to subsequently perform a pre-sequence digest of the plasmid preparation.
Protocols Used: Overnight Cultures
Results: Insert table values!
Next Steps: Looking at the pre-sequence digest it has been decided that sample 5 will be sent for sequencing. This will be done on the 29/6.
Summary
Correct insertion of OBP2A into pSB1C3 determined through sequencing.
Aim of Experiment: Sent sample 5 of the plasmid preparation that was done on the 24/6 for sequencing to determine if OBP2A has successfully ligated into pSB1C3.
Protocols Used:
Results: N/A
Next Steps: If sequencing comes back positive, then the next step will be to clone this gene insert into the two constituents of the two hybrid system.
Aim of Experiment: Sequencing came back positive for pSB1C3-OBP2A. Soan overnight culture of pSB1C3-OBP2A from the sequenced colony will be done overnight in preparation for plasmid preparation.
Protocols Used:Overnight Cultures
Results: N/A
Next Steps:Once the overnight culture has been given ample time to grow, the culture will then undergo a plasmid preparation.
Aim of Experiment: To produce a plasmid preparation of the sequenced pSB1C3-OBP2A from the overnight culture set up on the 30/6.
Protocols Used:Miniprep
Results: N/A
Insert plasmid concentration.Next Steps: the next step of experimentation is to use the plasmid preparation as a template for PCR using primer designed to separate the gene for use in the two hybrid system.
Summary
Ran PCR of OBP2A using primers for insertion of OBP2A into PUT18 and PT25.
Aim of Experiment: To PCR OBP2A for its insertion into the bacterial two hybris vectors; PUT18 and PT25.
Protocols Used: Transformation
Results: N/A
Next Steps:Tthe next stage of experimentation will be to gel extract and restrict the PCR gene fragments.
Summary
Removal of the signaling peptide from OBP2A and insertion of OBP2A into the biobrick vector pSB1C3.
Aim of Experiment: To perform a PCR on the plasmid preparation of pIDT-OBP2A produced last week, using primers that have been designed in a way as to remove the signalling peptide at the start of the OBP2A gene.
Protocols Used: PCR
Results: N/A
Next Steps: The next step will be to run the PCR product on a gel and perform a gel extraction on the part of the gel corresponding to the size of the OBP2A gene fragment.
