Uppsala-Sweden/14 August 2009

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--[[User:Karl.brune|Karl.brune]] 14:28, 14 August 2009 (UTC)
--[[User:Karl.brune|Karl.brune]] 14:28, 14 August 2009 (UTC)
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==Plasmid prep contiued==
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==Plasmid prep continued==
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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 ''m''M! I precipitated the DNA wizh ethanol and resuspen in 5 mM Tris-HCl.
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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 ''m''M! I precipitated the DNA with ethanol and resuspended it in 5 mM Tris-HCl.
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Left to do: Measure DNA koncentration for all the plasmid preps.
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Left to do: Measure DNA concentration for all the plasmid preps.
--[[User:Karl.brune|Karl.brune]] 14:28, 14 August 2009 (UTC)
--[[User:Karl.brune|Karl.brune]] 14:28, 14 August 2009 (UTC)
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==Plasmid Measurements==
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Anders made the measurements of the DNA concentration with 100 times dilutions.
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[[Media:Plasmidprep_080914.pdf]]
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--[[User:Karl.brune|Karl.brune]] 22:24, 14 August 2009 (UTC)
==Picking adh2 (Z) colonies==
==Picking adh2 (Z) colonies==
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--[[User:Karl.brune|Karl.brune]] 14:49, 14 August 2009 (UTC)
--[[User:Karl.brune|Karl.brune]] 14:49, 14 August 2009 (UTC)
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==Colongy PCR of new adh2 (Z)==
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==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..)
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..)
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==PCR of pirA==
==PCR of pirA==
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New primers for pirA according to the FEBS article.  
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New primers for pirA according to the [http://www.google.com/#hl=en&q=A+cyanobacterial+gene+encoding+an+ortholog+of+Pirin+is+induced+under+stress+conditions&aq=f&aqi=&fp=82e34627c46f57f4 FEBS article by Hihara].  
Gradient according to table
Gradient according to table
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#1 @ 43,2°C
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#2 @ 46,3°C
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# @ 43,2°C
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#3 @ 48,2°C
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# @ 46,2°C
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#c @ 60,7 (pirB control)
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# @ 48,3°C
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# @ 60,7 (pirB control)
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Performed with Phusion .
Performed with Phusion .
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--[[User:Karl.brune|Karl.brune]] 14:51, 14 August 2009 (UTC)
--[[User:Karl.brune|Karl.brune]] 14:51, 14 August 2009 (UTC)
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==Gel of PirA v2 Grad PCR==
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[[Image:20090814-pirAv2grad-labbeled.jpg]]
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--[[User:Karl.brune|Karl.brune]] 16:44, 15 August 2009 (UTC)
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==Gel of colony PCR==
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Here is the gel from the colony PCR.
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[[Image:20090814-colonypcr3-labelled.jpg]]
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--[[User:Karl.brune|Karl.brune]] 22:24, 14 August 2009 (UTC)

Latest revision as of 16:44, 15 August 2009




Contents

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 [http://www.google.com/#hl=en&q=A+cyanobacterial+gene+encoding+an+ortholog+of+Pirin+is+induced+under+stress+conditions&aq=f&aqi=&fp=82e34627c46f57f4 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

20090814-pirAv2grad-labbeled.jpg

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

Gel of colony PCR

Here is the gel from the colony PCR.

20090814-colonypcr3-labelled.jpg

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

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