Team:Groningen/Notebook/14 July 2009

From 2009.igem.org

Latest revision as of 12:19, 17 August 2009

Wet

GVP Cluster

www.sigmaaldrich.com

Plasmid Purification

Plasmid isolation was performed on the cultures of GVP and Terminator containing cells with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit".

• From each tube 5mL of culture was collected in a 2mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
• Cells were resuspended in 200μL Resuspension Solution by up and down pipetting.
• To the mixture 200μL Lysis Solution was added, mixed by inverting the cup, and stored at room temperature for 5 minutes.
• 350μL of Neutralisation Solution was added and the tubes inverted.
• Cell debri was pelleted by centrifugation at full speed for 10 min.
• Column was prepared by adding 500μL Column Preparation Solution and centrifuging for 1 min. full speed
• The lysate was transfered to the column and centrifuged for 1 min. full speed
• Column was washed with both washing solutions
• Plasmids were eluted with 30μL MQ and stored in the fridge

Concentration of Plasmids

The concentration of isolated plasmid was determined with the use of a nano-drop.

[http://partsregistry.org/wiki/index.php/Part:BBa_I750016 GVP] eluted in MQ

• 329.7 ng/μL
• 1.84 (260/280)
• 2.13 (260/230)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109] eluted in MQ

• 174.9 ng/μL
• 1.88 (260/280)
• 1.92 (260/230)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] eluted in MQ

• 155.3 ng/μL
• 1.85 (260/280)
• 1.79 (260/230)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106] eluted in MQ

• 219.4 ng/μL
• 1.86 (260/280)
• 1.93 (260/230)

Restriction of GVP and J23100/106/109 for assembly

A restriction was performed on the parts [http://partsregistry.org/wiki/index.php/Part:BBa_I750016 GVP], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106] as a first step in the construction of a new part containing the promoter and the GVP gene cluster.

Restriction mixture for GVP (fragment sizes: 6096 (GVP cluster), 3513)

• 10 μL GVP plasmid (88.2 ng/μL)
• 6μL MQ
• 2μL Fast digest buffer
• 1μL PstI fast digest enzyme
• 1μL XbaI fast digest enzyme

Restriction mixture for J23109/100 (fragment sizes: 2096 (promoter + plasmid backbone), 887)

• 10 μL promoter plasmid (61.4 ng/μL, 96.1 ng/μL)
• 6μL MQ
• 2μL Fast digest buffer
• 1μL PstI fast digest enzyme
• 1μL SpeI fast digest enzyme

- for J23106

• 8 μL promoter plasmid (55.4 ng/μL)
• 8μL MQ

Incubation for 30 min at 37°C.

The digests were run on a gel (1% agarose, TBE) and the bands containing the necessary fragments (6096, 2096) were excised.

Purification of fragments from agarose gel

Purification is done with the High pure PCR product purification kit (Roche).

• 300 μL Binding buffer added for every 100 mg agarose
• vortexed 30 s
• 10 min incubation in 70°C (every 3 min vortexed 10 s)
• 150 μL isopropanol added forevery 100 mg of agarose
• vortexed 15 s
• Contents pipetted into a filter tube
• Centrifugation 60 s max speed (14600 rpm), flowthrough is discarded
• 500 μL Wash buffer added, centrifugation 60 s max speed, flowthrough is discarded
• 200 μL Wash buffer added, centrifugation 60 s max speed, flowthrough is discarded
• 50 μL Elution buffer added, centrifugation 60 s max speed
• Flowthrough contains the product DNA

Concentrations of DNA

Determined with a Nanodrop.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109] eluted in Elution buffer

• 3.5 ng/μL
• 1.90 (260/280)
• 0.34 (260/230)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] eluted in Elution buffer

• 4.6 ng/μL
• 2.64 (260/280)
• 0.42 (260/230)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106] eluted in Elution buffer

• 2.5 ng/μL
• 2.10 (260/280)
• 0.07 (260/230)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 GVP (1)] eluted in Elution buffer

• 3.5 ng/μL
• 4.42 (260/280)
• 0.05 (260/230)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 GVP (2)] eluted in Elution buffer

• 5.2 ng/μL
• 2.26 (260/280)
• 0.07 (260/230)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 GVP (3)] eluted in Elution buffer

• 5.9 ng/μL
• 2.36 (260/280)
• 0.28 (260/230)

MQ was measured at the end: 1.8 ng/μL

Transporters

3nd PCR HmtA_Fw/HmtA_Rev We have no products on our gel but we do have ON cultures, we picked 3 for isolation.

Today we have the other primers as well. Since we are almost out of template DNA we preform colony PCR on the Colonies of the HmtA plate 3. With a dynazyme master mix (2 samples picked) and homebrew (2 samples picked). To see which ON culture to isolate plasmide from. and continue Cloning by mutating with mutation primer on the PCR product.

Vectors

Innoculated reaction tubes filled with 3 ml Ty or LB for overnight shaking 37°

• pSB3K3 3ml kan LB and Ty
• pSB1AC3 3ml amp LB and Ty
• BBa_J23100 3ml amp LB and Ty
• BBa_J23106 3ml amp LB and Ty
• BBa_J23109 3ml amp LB and Ty

Dry

The arsenic accumulation model was worked out to the point where it supports pretty much all the kinds of operators we're interested in (at least the equilibrium model), and slightly more realistic parameters were used in the Simbiology model. The equilibrium calculator on the Wiki uses [http://en.wikipedia.org/wiki/Fixed_point_iteration fixed point iteration] to compute the equilibrium, so far this has proven to be quite fast and accurate (in addition it is full of sanity checks). And nicely enough the calculator on the Wiki so far agrees with the Simbiology model!