Uppsala-Sweden/14 August 2009

Transformation of Adh2 z, evaluation of competence
continued since yesterday

So, it's official, our competent cells are really quite incompetent! Tested against the new bath of competent DH5alfa which got 220 transformations (100*10^6 / µg pUC 19)our perfored extremly bad at around 20 transformations.

We will make new CCMB80 (unauotclaved this time) and also consider to switch cells to TOP10 instead.

--Karl.brune 14:28, 14 August 2009 (UTC)

Plasmid prep continued
Yesterday we performed a plasmid prep on all transformed genes. Today I did the same for the B0034 RBS. This with a small screw up as I accedentilly eluted with 1 M Tris-HCl as opposed to 5 mM! I precipitated the DNA with ethanol and resuspended it in 5 mM Tris-HCl.

Left to do: Measure DNA concentration for all the plasmid preps.

--Karl.brune 14:28, 14 August 2009 (UTC)

Plasmid Measurements
Anders made the measurements of the DNA concentration with 100 times dilutions.

[[Media:Plasmidprep_080914.pdf]]

--Karl.brune 22:24, 14 August 2009 (UTC)

Picking adh2 (Z) colonies
I inoculated 15 adh2 (Z) colonies in 200µl of LB with 50µg/ml amp in 1,5ml eppendorfs. Picked them with a 10µl tip and dropped them in the eppendorf. Inoculated at 37°C and 250 rpm. Colony PCR and gel coming soon. (further down)

--Karl.brune 14:49, 14 August 2009 (UTC)

Colony PCR of new adh2 (Z) colonies
Colongy PCR of adh2 (Z) (VF2 and VR). Final volume 10µl with Taq Polymerase at 50°C annealing temperature. Performed on PCR machine #2 (Eppendorf oldschool grad, real spec coming..)

--Karl.brune 14:49, 14 August 2009 (UTC)

PCR of pirA
New primers for pirA according to the FEBS article by Hihara. Gradient according to table


 * 1) @ 43,2°C
 * 2) @ 46,2°C
 * 3) @ 48,3°C
 * 4) @ 60,7 (pirB control)

Performed with Phusion.

Gel coming.

--Karl.brune 14:51, 14 August 2009 (UTC)

Gel of PirA v2 Grad PCR


--Karl.brune 16:44, 15 August 2009 (UTC)

Gel of colony PCR
Here is the gel from the colony PCR.



--Karl.brune 22:24, 14 August 2009 (UTC)