Team:Groningen/Notebook/13 July 2009

From 2009.igem.org

Latest revision as of 12:24, 16 July 2009

Wet

GVP Cluster

Restriction for Assembly

The vector containing GVP cluster was cut with EcoRI and XbaI to create correct ends.

• 6μL MQ
• 10μL plasmid in MQ
• 2μL Fast digest buffer
• 1μL EcoRI fast digest enzyme
• 1μL XbaI fast digest enzyme

The vectors containing promotors [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] were cut with EcoRI and SpeI.

• 0μL MQ
• 16μL plasmid in MQ ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106])
• 2μL Fast digest buffer
• 1μL EcoRI fast digest enzyme
• 1μL SpeI fast digest enzyme

-

• 6μL MQ
• 10μL plasmid in MQ ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100])
• 2μL Fast digest buffer
• 1μL EcoRI fast digest enzyme
• 1μL SpeI fast digest enzyme

The cups were incubated in a heat block at 37°C for 15 min. followed by adding 4μL 6x loading buffer for gel electroforesis.

Gel electroforesis

25μL of each sample was loaded on a 1% agarose gel with EtBr and a 1kb ladder was used (see picture).

→ From left to right: 1kb Marker, Empty Slot, [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109], Empty Slot, [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100], Empty Slot, [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106], Empty Slot, GVP, Empty Slot

→ The promotor parts (50bp) were not visible on gel after a run at 100V for 25 min. A second gel run for 10 min. at 100V showed the same result for promotor [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100], but had a vage outline of the correct fragment size (gel not shown). Purification of this part of the gel would yield a far too low concentration, and was therefor not attempted.

Gel Purification of GVP

A standard kit for PCR-product purification was used for gel purification

• 120mg of gel containing the desired fragment was dissolved in 500μL binding buffer by heating to 60°C for 10 min.
• the column was prepared with 500μL Preparation Solution and centrifuged for 1 min.
• dissolved gel was transfered to the column and centrifuged for 1 min.
• column was washed with 750μL Wash Solution and centrifuged twice for 1 min.
• 15μL MQ was applied to the column, incubated for 3 min. at room temperature and centrifuged for 1 min. at full speed.

Concentration of GVP-vector

The concentration of isolated cut vector of GVP with EcoRI and XbaI was determined with the use of a nano-drop.

GVP-vector eluted in MQ

• 4.3 ng/μL
• 1.42 (260/280)
• 0.60 (260/230)

→ After the low concentration was determined, the cup was disposed of because a higher concentration is required!

Inoculation of TY-medium

TY-medium (5mL) was inoculated with a tip of glycerol stocks:

• GVP no.1
• [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109]
• [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100]
• [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106]

and 5μL ampicilin (1000x). The tubes were placed in a 37°C shaker for over night incubation at 200 rpm.

Transporters

2nd PCR HmtA Mut1/Mut2

The gel shows a small band in lane with Mg&K Mastermix. This indicates low yields, next large fragment >5kb should be done with Phusion mastermix. And if we have time we should play a bit with mastermix contend to find the optimal mixture.

3nd PCR HmtA_Fw/HmtA_Rev

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.

Dry

Reaction rates were set up for arsenic accumulation and a Simbiology model created (see SVN). Quite some time was spent on looking for some of the missing parameters. No specific values were found, but perhaps we could estimate some using software?

Furthermore a start was made with an analysis of accumulation under the equilibrium assumption. This makes more sense than a "normal" model if you just want to know the amount of arsenic accumulated while varying a parameter for example.