Team:Groningen/Notebook/17 July 2009

From 2009.igem.org

Revision as of 14:32, 17 July 2009

Wet

GVP Cluster

Discussion:

All colonies (transformants vector + promoter ligated with gvp) and o/n culture became red, so probably RFP is still in there... What might be the problem? The vector with promoter self-ligated due to uncomplete digestion (done 15 July 2009) leading to the following fragments:

• vector+RFP linear
• vector
• RFP

When the upper fragment (~2kb) is contaminated with linear vector this would lead high efficiency self-ligation instead of ligating gvp with the emtpy vector.

→TODO Therefore the restriction/purification/ligation will be redone and selection of non-red colonies will be made!

→DONE Also plasmid from the o/n cultures will be purified and analysed by restriction analysis.

• For plasmid isolation the Sigma Plasmid isolation kit was used (eluted in 50ul MQ).
• DNA concentrations:

• Restriction analysis with SpeI and PstI to find insert length.
  • Expected length of with / with RFP insert: 2096bp(promoter + plasmid backbone), 887bp(RFP)
  • Expected length of with gvp insert: 2096bp(promoter + plasmid backbone),6096bp(gvp)
  • Expected length of with gpv insert: 3513(plasmid backbone), 6096 (GVP cluster)

Restriction mixture for J23100+gvp and GVP

• 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

10ul was loaded on a 1%TBE agarose gel and ran for 30 @ 100V (V const)

→From left to right: 1kb marker, gvp-promoter () construct nr 9, nr 10, gvp in , promoter ()

So the assumption that the vector with the promoter self-ligated was right, as the gvp-promoter construct has the same bands as the promoter has and the size of the bands are as expected for a RFP insert.

Transporters

Because of the negative results we will try to get positive results with an other polymerase enzyme; Phusion. And try the cloning again as planned. PCR1 Fw,mut1RC should give product of 1153bp and PCR2 Rev,mut2RC a 261bp size product. Further more we will test a PCR with pBAD 24 Fw,rev to get smaller templates.

Metal Accumulation

Vectors

Dry