Uppsala-Sweden/13 August 2009

Transformation of ADH2 from z.mobilis, continued
Unfortunatly we only got two (!!) colonies from Adh2 z. These are most likely to be contamination as one was found on 50 µl olate and on on the 550 µl plate, we will still evaluate them with a colony PCR however.

The control pUC 19 yielded 59 colonies at the 20 µl plate giving a transformation efficiency of 70,8*10^6 cfu/µg pUC19. This compared to what is considered good transformation efficiency by TOP10 chemically competent cells, 500 - 5000 *10^6 cfu/µg pUC19.

Becouse of this we decided to test the competence of our cells against another batch of the same type of cells done here in the lab. This is done today and incubated over nigth so we will have the answer tomorrow.

If the other cells are as crappy we will change to TOP10 cells also available. The most likely couse of failure is that we autoclaved the CCMB80 buffer, against protocol, with coused a reddish brown, likely manganese, precipitation.

When transforming this test we also throw in some Adh2 z to see if we get lucky. Even with very bad cells (and ours are merly not very good it seems) there should be some colonies of Adh2 z.We investigate the couse of this below.

--Anders 15:44, 13 August 2009 (UTC)

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

--Karl.brune 18:09, 13 August 2009 (UTC)

Glycerol Stocks of pSB1A3 (2), pSB1AC3 (2), pSB1AK3 (2), pPetE (4), pirB (2), pdc (1), pUC19 (1)
21,75% Glycerol Stocks of pSB1A3 (2), pSB1AC3 (2), pSB1AK3 (2), pPetE (4), pirB (2), pdc (1), pUC19 (1) were performed on overnight cultures. 800µl culture + 200µl 87% sterile glycerol. Stored in rack 10 in the iGEM @-80°C box in the ultrafreezer.

--Karl.brune 18:09, 13 August 2009 (UTC)

Miniprep of pSB1A3 (2), pSB1AC3 (2), pSB1AK3 (2), pPetE (4), pirB (2), pdc (1), pUC19
Miniprep of pSB1A3 (2), pSB1AC3 (2), pSB1AK3 (2), pPetE (4), pirB (2), pdc (1), pUC19 was performed with 5ml overnight culture each with Sigma GenElute™ Plasmid Miniprep Kit. Final eluate is 5mM Tris-HCl (100µl). Concentration will be measured tomorrow and supplied here as well. Currently stored in the iGEM freezer (-20°C) rack2, taped together. (Keep in order!!!)

--Karl.brune 18:04, 13 August 2009 (UTC)

Colongy PCR adh2 (Z) with adh2 (Z) ligates
Colongy PCR of adh2 (Z) (VF2 and VR) adh2 (Z) ligates (VF2 and VR) from the last two times (not including the latest ligation). Final volume 10µl with Taq Polymerase at 50°C annealing temperature. Performed on PCR machine #2 (Eppendorf oldschool grad, real spec coming..)

--Karl.brune 18:04, 13 August 2009 (UTC)

Gel on colony PCR, PCR'd ligate products and non-PCR ligates
1% Agarose Gel loaded with 2.2µl+0.8Loading Dye at 160V for 30min with 1kb ladder (Fermentas 1kb Gene Ruler). On colony PCR products, PCR'd ligates products and on non-PCR ligates (adh2 ligates)#



--Karl.brune 18:04, 13 August 2009 (UTC)

New primer ordered for adh2(Y)
I ordered new primers for adh2(Y), this time including the second stop codon. ;)

--Karl.brune 18:04, 13 August 2009 (UTC)