Team:UGA-Georgia/NotebookE.coli

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= Notebook =
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= E. coli Lab =
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== '''Methanococcus Lab''' ==
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Instructor: ZHE LYU
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'''February 2013'''
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-Training on anaerobic skills, i.e. use of anaerobic glassware, gassing chamber,
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anaerobic chamber, etc.
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'''March 2013'''
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-Purification of GS, AT and GS+AT via revival of frozen stocks and plating of sub-cultures.
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'''April 2013'''
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-Further continuation of purification of GS, AT and GS+AT via picking colonies, creating sub-cultures and plating.
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-Test of Puromycin strength.
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-Creation of frozen stocks of purified GS, AT and GS+AT transformants.
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'''June 2013'''
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-Extraction of Geraniol from extra-cellular and intra-cellular content of samples and preparation for GC/MS evaluation
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-Sequencing of pAW50-mCherry vector
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-Analysis of pAW50-mCherry sequence
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-Transformation of pAW50-mCherry vector into Methanococcus
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-Testing Fluorescence of pAW50-mCherry
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-Purification of pAW50-mCherry cultures via plating of transformants, and creating sub-cultures of colonies picked
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-Creation of frozen stocks of pAW50-mCherry in Methanococcus
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'''July 2013'''
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-Creation and analysis of the "killing & inhibiting" experiment where we test the maximum amount of product a 5ml culture of Methanococcus can tolerate in cultures with high OD (killing of grown cells) and low OD (inhibiting of growth).
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-Innovation of an adapter to allow the use of syringe needles on micropipettes.
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-Expanded upon the original extraction protocol for higher efficiency of extraction of geraniol from Methanococcus cultures.
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- GC/MS Evaluation and analysis (see results tab)
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'''August 2013'''
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-Revival and PCR of all GS and AT frozen stocks to confirm insert.
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--Extraction of Geraniol from extra-cellular and intra-cellular content of all GS frozen stocks and preparation for GC/MS evaluation
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'''September 2013'''
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-Transformation of V4 into Methanococcus
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-Testing Fluorescence of V4
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-Purification of V4 cultures via plating of transformants, and creating sub-cultures of colonies picked
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-Creation of frozen stocks of V4 in Methanococcus
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-GC/MS Evaluation and analysis (see results tab)
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== '''E. coli Lab''' ==
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Instructor: Rachit Jain
Instructor: Rachit Jain
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'''March 2013'''
'''March 2013'''
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-PCR of GS gene
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-PCR of GS gene with histidine tag (histag).
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-Cloning of GS gene into pAW-50.
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-Cloning of GS-histag gene into pAW-50.
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-Transformation of pAW-50 GS into species XL1-Blue. Transformed species spread onto ampicillin plates
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-Transformation of pAW-50 GS-histag into species XL1-Blue. Transformed species spread onto ampicillin plates
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-Verification of GS transformants via digestion and gel verification
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-Verification of GS-histag transformants via digestion and gel verification

Latest revision as of 23:36, 25 September 2013

E. coli Lab

Instructor: Rachit Jain


February 2013

-Training on PCR and heat shock transformation


March 2013

-PCR of GS gene with histidine tag (histag).

-Cloning of GS-histag gene into pAW-50.

-Transformation of pAW-50 GS-histag into species XL1-Blue. Transformed species spread onto ampicillin plates

-Verification of GS-histag transformants via digestion and gel verification


April 2013

-Colonies for GS, AT, and GS+AT were selected from Methanococcus and corresponding genes were extracted. PCR and gel verification of GS, AT, and GS+AT genes. Results concluded successful in locating vectors from Methanococcus.

-PCR and gel verification of mCherry gene

-Cloning of mCherry gene into pAW-50 vector

-Verification of cloning via digestion and gel verification. After positive verification, ligation of digested products and heat shock transformation into XL1-Blue. XL1-Blue transformants spread onto ampicillin plates.

-XL1-Blue colonies picked and screened. Purification of pAW-50 mCherry from colonies, and stored in -20°C freezer.


June 2013

-Transformation of pAW-50 mCherry into XL1-Blue and creation of stock

-PCR of two mCherry sequences: one without RBS sequence (V2) and one containing RBS sequence (V4). Gel Verification of tools 1 and 2.

-Digestion and cloning of V2 and V4 into pAW-50 mCherry vector


July 2013

-Purification of digested vector, ligation, and transformation of vector into XL1-Blue. Transformants plated onto ampicillin plates.

-V2 and V4 colonies extracted and screened. Permanent stocks of V2B and V4B stored in -80°C freezer.

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