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.

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

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.

Aim of Experiment: To perform a gel extraction and then subsequently a restriction of OBP2A from the PCR produced on the 17/7 that used primers designed for the insertion ofOBP2A into the vectors for the bacterial two hybrid system, pUT18 and pT25.

Protocols Used: Gel extraction Restriction

Results: N/A

Next Steps: If the gel extraction and restriction is successful, then the next step will be to ligate these restrictions into the bacterial two hybrid vectors, pUT18 and pT25.

Aim of Experiment: To perform a ligation and then subsequently a transformation of OBP2A into the vectors for the bacterial two hybrid system, pUT18 and pT25. Whilst using JM110 E.coli cells as a chassis for transformation.

Protocols Used: Ligation Transfromation

Results: N/A

Next Steps: If transformation is successful and positive colonies form on the antibiotic plates, then the next step will be to grow overnight colonies in preparation for purification and sequencing.

Aim of Experiment: As the transformation from the 21/7 failed - no positive colonies grew on the antibiotic plates. A re-ligation and transformation of OBP2A into pUT18 and pT25 was done.

Protocols Used: ligation Transformation

Results: N/A

Next Steps: If transformation is successful and positive colonies form on the antibiotic plates, then the next step will be to grow overnight colonies in preparation for purification and sequencing.

Aim of Experiment: Although the transformation of OBP2A into pT25 was unsuccessful, the transformation of OBP2A into pUT18 did produce positive colonies. These colonies were grown overnight in preparation for Miniprep.

Protocols Used: Overnight Culture p>

Results: N/A

Next Steps: Once the cells have grown overnight they will undergo plasmid preparation for subsequent sequencing.

Aim of Experiment: As he first attempt of OBP2A insertion into pT25 was unsuccessful, a further PCR and gel extraction was done.

Protocols Used: PCR

Results: N/A

Next Steps: The next stage of experimentation will be to restrict OBP2A for subsequent ligation into pT25.

Aim of Experiment: A restriction of OBP2A for PT25 was performed. Sequencing came back positive for pUT18-OBP2A, so pUT18-OBP2A can now be implemented in the bacterial two hybrid system.

Protocols Used: Restriction p>

Results: N/A

Next Steps: The next stage of experimentation will be to ligate OBP2A into the pT25 vector.

Aim of Experiment: As the restriction was done yesterday a ligation and transformation of OBP2A into the bacterial two hybrid vector pT25 was done today.

Protocols Used: ligationTransformation

Results: N/A

Next Steps: If transformation is successful and positive colonies form on the antibiotic plates, then the next step will be to grow overnight cultures of those colonies in preparation for purification and sequencing.

Aim of Experiment: The transformation of OBP2A into pT25 produced positive colonies, so overnight cultures of these colonies were done today.

Protocols Used: Overnight Culture p>

Results: N/A

Next Steps: Once the cells have grown overnight they will undergo plasmid preparation for subsequent sequencing.

Aim of Experiment: The overnight cultures of pT25-OBP2Aunderwent plasmid preparation and then a subsequent pre-sequence digest before being sent for sequencing.

Protocols Used: Miniprep Pre-sequence digest p>

Results: N/A

Next Steps: If sequencing comes back positive for pT25-OBP2A then this can now be implemented into the bacterial two hybrid system. The results from the sequencing should arrive early on the 3/8.

Aim of Experiment: In order to gauge the interaction between the two separate parts of my protein both bacterial two hybrid plasmids must be transformed into the same cell. In this case the transformation is being done with the BTH101 E.coli strain.This is the first of two transformations.