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	= Notebook =		= Notebook =
-	'''Methanococcus Lab'''
-	'''Tasks completed before 2013/02/20'''	+	== '''Methanococcus Lab''' ==
-	-Training on anaerobic skills, i.e. use of anaerobic glassware, gassing chamber,	+	Instructor: ZHE LYU
-	anaerobic chamber, etc.	+
-		+
-	-Transferred and grew Methanococcus transformants with the geraniol synthase	+
-	gene encoded in the plasmid that was stored via glycerol stock from Summer	+
-	2012. Transformants containing only the pAW42 vector (control) were also	+
-	transferred and grown.	+
-		+
-	-Cell samples from the cultures listed above were harvested, resuspended in	+
-	buffer and stored at -20C for running SDS in near future.	+
-		+
-	-Cell samples from cultures listed two above were harvested, flash-frozen and	+
-	stored at -80C formRNA extraction in the near future.	+
-		+
-	-Both liquid and solid anaerobic medium was prepared for future use. Refer to	+
-	Appendix of protocols, I Preparation of Complex Medium for Methanococci	+
-	(McC).	+
-		+
-	'''2013/2/20'''	+
-		+
-	-Further preparation of media tubes via inoculation of Puromycin and	+
-	pressurization to 40 psi using H2/CO2 gas. Stored at -20C.	+
-		+
-	-Preparation of samples for SDS gel.	+
-		+
-	-Vector (control) samples made at 1x dilution and 5x dilution	+
-		+
-	-Geranyl Synthase (GS) plasmid samples made at 1x and 5x dilution	+
-		+
-	-An SDS gel was run using both dilutions of vector and GS samples, along with a	+
-	protein marker.	+
-		+
-	'''2013/2/26'''	+
-		+
-	-Four media tubes were treated with 0.1 mL Na2S solution each. Two of the tubes	+
-	were used for inoculation of GS transformant, and the other two were used for	+
-	inoculation of GS + AT (geraniol acetyltransferase) transformant.	+
-		+
-	-200x dilution of Puromycin solution was created and properly dispensed.	+
-		+
-	-SDS gel from 2013/2/20 was imaged. Gel image quality was lacking, so it was	+
-	decided to perform a second gel.	+
-		+
-	'''2013/2/27'''	+
-		+
-	-Reflecting upon first SDS gel, new sample dilutions were created in aim of	+
-	creating an easier to read gel. 2x dilution sample of GS was created and leftover	+
-	5x GS sample from first gel was also used.  4x, 6x and 8x dilutions of vector	+
-	samples were created, and leftover 5x dilution vector samples from first gel were	+
-	also used. Lastly, a protein marker ladder was used for size reference.	+
-		+
-	-Gel was run, stained, de-stained and imaged (see below). No evident expression	+
-	of GS protein. Next verification of this will be to run an mRNA extract for	+
-	conclusive evidence of GS gene being transcribed	+
		+	'''February 2013'''
		+	-Training on anaerobic skills, i.e. use of anaerobic glassware, gassing chamber,
		+	anaerobic chamber, etc.
		+	'''March 2013'''
-	[[[[[[[[[[[[[[[[[[[[Fig 1. ]]]]]]]]]]]]]]]]]]]	+	-Purification of GS, AT and GS+AT via revival of frozen stocks and plating of sub-cultures.
		+	'''April 2013'''
		+	-Further continuation of purification of GS, AT and GS+AT via picking colonies, creating sub-cultures and plating.
		+	-Test of Puromycin strength.
		+	-Creation of frozen stocks of purified GS, AT and GS+AT transformants.
-	'''2013/03/05'''	+	'''June 2013'''
-	-6 tubes of McC media created, added Na2S (0.1mL 2.5%) and Puromycin (0.5mL of	+	-Extraction of Geraniol from extra-cellular and intra-cellular content of samples and preparation for GC/MS evaluation
-	10x)	+
-	-Inoculated vector strain Methanococcus into two tubes and GS + AT into other two	+	-Sequencing of pAW50-mCherry vector
-	tubes.	+
		+	-Analysis of pAW50-mCherry sequence
-	'''2013/03/06'''	+	-Transformation of pAW50-mCherry vector into Methanococcus
-	-Solid McC media was autoclaved so gel would melt, so we could add Na2S and	+	-Testing Fluorescence of pAW50-mCherry
-	Puromycin to all solid media bottles for plating. Puromycin was added to all but two	+
-	bottles. These two bottles will be used as control for Puromycin.	+
-	-GS and AT cell cultures were measured for their optical density (OD). A cultures OD	+	-Purification of pAW50-mCherry cultures via plating of transformants, and creating sub-cultures of colonies picked
-	can tell us how well its growth and reproduction is. There is a direct correlation between	+
-	OD and cells/mL, so we will use this to determine how much we must dilute to achieve	+
-	around 150 cells/ml for optimal plating.  Appropriate dilutions were measured and	+
-	created.  These samples were added to solid media bottles is various quantities (0.3mL,	+
-	0.6mL and 0.9mL), allowed to rest on the agar media for half an hour, then placed	+
-	vertically into a 37C incubation room.	+
-	'''2013/03/19'''	+	-Creation of frozen stocks of pAW50-mCherry in Methanococcus
-	-4 media tubes were prepared with 0.1 mL of 2.5% Na2S solution. These tubes
-	were then pressurized to 40 psi using H2/CO2 gas.
-	-Puromycin solution was prepared: 7.5mg of 200x Puromycin was added to 30	+	'''July 2013'''
-	mL of de-ionized water and sparged in nitrogen gas. The 30 mL was then	+
-	transferred to 6 tubes, each with 5 mL of 100x Puromycin.	+
-	-6 solid media plates were prepared for future AT Methanococcus. The 6 bottles	+	-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).
-	were autoclaved, and treated with 0.1 mL 100x Puromycin and 0.2 mL 2.5%	+
-	Na2S solution each.	+
-	-1 GS (CK or control) colony, and 1 GS colony was slected and inoculated to	+	-Innovation of an adapter to allow the use of syringe needles on micropipettes.
-	media containing 0.1 mL 2.5% Na2S. Note that these 2 colonies were not well	+
-	separated.  The 2 cultures were added to a 37C incubator.	+
-	'''2013/03/20'''	+	-Expanded upon the original extraction protocol for higher efficiency of extraction of geraniol from Methanococcus cultures.
-	-200 mL liquid media was created, dispensed into 40 tubes, stoppered and capped,	+	- GC/MS Evaluation and analysis (see results tab)
-	pressurized to 15 psi using nitrogen and carbon dioxide, autoclaved and placed in the	+
-	anaerobic chamber.	+
-	-Frozen transformants of AT and GS+AT were placed into two properly treated media
-	tubes and placed into 37C incubator.
-	-Various appropriate dilutions of GS and AT cultures were created relative to OD
-	readings as described in the process from 2013/03/06.
-	-These dilutions were inoculated into 8 solid media bottles for plating, and stored at 37C	+	'''August 2013'''
-	incubation room.	+
-	'''2013/03/26'''	+	-Revival and PCR of all GS and AT frozen stocks to confirm insert.
-		+
-	-8 media tubes were treated with 0.1 mL of 2.5% Na2S solution, 0.05 mL of 100x	+
-	Puromycin solution and pressurized to 40 psi with H2 and CO2. Two of these tubes were	+
-	inoculated with 0.2 mL of AT transformant , while 2 other tubes were inoculated with 0.2	+
-	mL of AT+GS transformant.  The remaining 4 tubes were inoculated with colonies that	+
-	formed from the GS transformant after Puromycin purification.	+
-	-6 solid media bottles were autoclaved, treated with 0.2 mL Na2S and treated with 0.1	+	--Extraction of Geraniol from extra-cellular and intra-cellular content of all GS frozen stocks and preparation for GC/MS evaluation
-	mL of Puromycin for future plating.	+
-		+
-	'''2013/03/27'''	+
-		+
-	-Re-pressurize H2/CO2 culture tubes to 40 psi	+
-		+
-	-Place 1 mL of 60% glycerol/40% media solution into 20 three-mL serum bottles. These	+
-	20 bottles were stoppered and capped for future frozen culture stocks.	+
-	-Check OD of cultures, and decided they were not sufficient enough for proper plating.
-	'''2013/03/28'''	+	'''September 2013'''
-	-Re-pressurize H2/CO2 cultures tubes to 40 psi.	+	-Transformation of V4 into Methanococcus
-	-Measure OD of cultures tubes hoping much better growth since 2013/03/27. The OD	+	-Testing Fluorescence of V4
-	measurements provided results satisfactory for plating.	+
-	-Various appropriate dilutions of AT and GS+AT cultures were created relative to OD	+	-Purification of V4 cultures via plating of transformants, and creating sub-cultures of colonies picked
-	readings as described in the process from 2013/03/06.	+
-	-These diluted samples were inoculated into 6 solid media bottles for plating and plates	+	-Creation of frozen stocks of V4 in Methanococcus
-	were placed into 37C incubation room.	+
-	-3 autoclaved glycerol stock bottles made 2013/03/27 were used to create frozen culture	+	-GC/MS Evaluation and analysis (see results tab)
-	stocks of biological replicates of GS. Namely, GS-1, GS-2 and GS-3 which were derived	+
-	from 3 different GS culture tubes. These 3 GS-inoculated glycerol stock bottles were	+
-	placed in -80C freezer.	+
-	'''2013/04/02'''	+	== '''E. coli Lab''' ==
-		+
-	-400 mL of liquid media was created.	+
-	'''2013/04/03'''	+	Instructor: Rachit Jain
-	-Liquid media created 2013/04/02 was dispensed into 40 tubes, stoppered and capped. All
-	tubes were pressurized to 15 psi using nitrogen and carbon dioxide then autoclaved.
-	-Agar was weighed out for 20 solid media bottles. Liquid media was then dispensed to	+	'''February 2013'''
-	these solid media bottles in addition to the agar. These bottles were stoppered, capped,	+
-	pressurized and autoclaved like the previously mentioned 40 tubes.	+
-	-All tubes and bottles were moved to anaerobic chamber.	+	-Training on PCR and heat shock transformation
-	-Colonies were picked from an AT and AT+GS plate. Four colonies were taken from
-	each plate and inoculated into respective media tubes.
-	-Samples from the GS-1 and GS-2 frozen stock cultures were added to respective media	+	'''March 2013'''
-	tubes to test if the previously created frozen stock cultures are actually viable.	+
-		+
-	-The 10 tubes including the AT colonies, AT+GS colonies, and frozen stock samples	+
-	were added to an incubator for growth.	+
-	'''2013/04/09'''	+	-PCR of GS gene
-	-1 mL of two different GS cultures (labeled GSF-1 and GSF-2) were inoculated in a 1 mL	+	-Cloning of GS gene into pAW-50.
-	media/glycerol solution and stored at -80C for future use as a frozen stock culture.	+
-	-6 agar bottles were autoclaved, treated with 0.1 mL of 100x Puromycin and 0.2 mL	+	-Transformation of pAW-50 GS into species XL1-Blue. Transformed species spread onto ampicillin plates
-	2.5% Na2S. These bottles will be used for future plating.	+
-	-A test of Puromycin strength was started. 2 media tubes were used, one with 0.05 mL of	+	-Verification of GS transformants via digestion and gel verification
-	Puromycin and 0.1 mL Na2S, while the other was not treated with anything (to serve as a	+
-	control). From there, 0.2 mL of Methanococcus was added to each tube, and the tubes	+
-	will be compared for Archaeal growth in the future.	+
-	'''2013/04/10'''
-	-Measure OD for AT and AT+GS cultures	+	'''April 2013'''
-		+
-	-Various appropriate dilutions of AT and GS+AT cultures were created relative to OD	+
-	readings as described in the process from 2013/03/06.	+
-	-Make three plates for AT and three plates for AT+GS	+	-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.
-	-2 technical replicates at 10^-6 dilution at 0.6mL and 0.9mL per plate (using the	+	-PCR and gel verification of mCherry gene
-	tube with the higher OD measurement).	+
-	-1 biological replicate at 10^-5 dilution at 0.6mL (lower of the tubes OD	+	-Cloning of mCherry gene into pAW-50 vector
-	measurements).	+
-	-Create 9 glycerol stock serum bottles as described in 2013/03/27 for future frozen	+	-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.
-	stocks. 1 mLof glycerol was added, the bottles were then stoppered, capped, autoclaved	+
-	and moved into the anaerobic chamber.	+
-	'''2013/04/16'''	+	-XL1-Blue colonies picked and screened. Purification of pAW-50 mCherry from colonies, and stored in -20°C freezer.
-	-The two media tubes containing Methanococcus from 2013/04/09 were measured for
-	OD values. The Na2S + Puromycin containing media had on OD of 0.049A and the
-	control has an OD of 0.795. This data suggests that Puromycin is indeed still an effective
-	antibiotic.
-	-200 mL of liquid media was prepared.	+	'''June 2013'''
-	'''2013/04/17'''	+	-Transformation of pAW-50 mCherry into XL1-Blue and creation of stock
-	-Liquid media created 2013/04/16 was dispensed, stoppered, capped, pressurized to 15	+	-PCR of two mCherry sequences: one without RBS sequence (V2) and one containing RBS sequence (V4). Gel Verification of tools 1 and 2.
-	psi using nitrogen and carbon dioxide, autoclaved and placed back into the anaerobic	+
-	chamber.	+
-	-1 plate of AT+GS and 1 plate of AT samples created 2013/04/10 were used for creating	+	-Digestion and cloning of V2 and V4 into pAW-50 mCherry vector
-	cultures.	+
-	-Four colonies from each plate were inoculated into respective media tubes for plating,
-	and these plates were added to the incubator for growth.
-	2013/04/19	+	'''July 2013'''
-	-Colonies were picked from the plates created 2013/04/17 and inoculated into media	+
-	tubes. These tubes were added to a 37C incubator for growth. The success of these	+
-	colonies ends the purification process for all of our vectors.	+
		+	-Purification of digested vector, ligation, and transformation of vector into XL1-Blue. Transformants plated onto ampicillin plates.
-	[[[[[SUMMER SEMESTER]]]]]]]]]]]]]	+	-V2 and V4 colonies extracted and screened. Permanent stocks of V2B and V4B stored in -80°C freezer.

---

Latest revision as of 23:26, 22 September 2013

Notebook

Methanococcus Lab

Instructor: ZHE LYU

February 2013

-Training on anaerobic skills, i.e. use of anaerobic glassware, gassing chamber, anaerobic chamber, etc.

March 2013

-Purification of GS, AT and GS+AT via revival of frozen stocks and plating of sub-cultures.

April 2013

-Further continuation of purification of GS, AT and GS+AT via picking colonies, creating sub-cultures and plating.

-Test of Puromycin strength.

-Creation of frozen stocks of purified GS, AT and GS+AT transformants.

June 2013

-Extraction of Geraniol from extra-cellular and intra-cellular content of samples and preparation for GC/MS evaluation

-Sequencing of pAW50-mCherry vector

-Analysis of pAW50-mCherry sequence

-Transformation of pAW50-mCherry vector into Methanococcus

-Testing Fluorescence of pAW50-mCherry

-Purification of pAW50-mCherry cultures via plating of transformants, and creating sub-cultures of colonies picked

-Creation of frozen stocks of pAW50-mCherry in Methanococcus

July 2013

-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).

-Innovation of an adapter to allow the use of syringe needles on micropipettes.

-Expanded upon the original extraction protocol for higher efficiency of extraction of geraniol from Methanococcus cultures.

- GC/MS Evaluation and analysis (see results tab)

August 2013

-Revival and PCR of all GS and AT frozen stocks to confirm insert.

--Extraction of Geraniol from extra-cellular and intra-cellular content of all GS frozen stocks and preparation for GC/MS evaluation

September 2013

-Transformation of V4 into Methanococcus

-Testing Fluorescence of V4

-Purification of V4 cultures via plating of transformants, and creating sub-cultures of colonies picked

-Creation of frozen stocks of V4 in Methanococcus

-GC/MS Evaluation and analysis (see results tab)

E. coli Lab

Instructor: Rachit Jain

February 2013

-Training on PCR and heat shock transformation

March 2013

-PCR of GS gene

-Cloning of GS gene into pAW-50.

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

-Verification of GS 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.