Uppsala-Sweden/10 August 2009

Glycerol Stocks for pSB1A3, pSB1AC3 and pSB1AK3
Glycerol Stocks for pSB1A3, pSB1AC3 and pSB1AK3 were prepared with ON cultures. 800µl culture + 200µl 87% sterile gylcerol -> 21,75% Glycerol Stocks stored in rack 10, iGEM @-80°C

--Karl.brune 12:52, 10 August 2009 (UTC)

Colony PCR with subsequent gel
Colony PCR was performed. With VF2 and VR primers.

Furthermore the ligates of non-transforming vector-insert mixes were run together on on a gel. Control was pirB PCR product.



Digestion of adh2(Z), pirB, pPetB, pdc
Digestion was performed using EcoRI and PstI from Fermentas with this protocol (Protocol for Fast Digestion of Unpurified PCR Products with FastDigest® Enzymes))

The calculated amounts for the digestion are written down here : [[Media:Digestion_090810.xls]]

Purification of adh2(Z), pirB, pPetB, pdc
Purification of the digestion mix was performed with Nucleo Spin® Extract 2 PCR cleanup protocol from Clontech Purified product (20µl) in 1.5ml eppendorfs.

DNA Measurements
DNA concentration were measured for subsequent ligation.

[[Media:Afterpurification 090810.pdf]]

Please state for which dilution this measurment was taken! --Anders 15:20, 11 August 2009 (UTC)

Edit: at 100 times dilution.. (cheers! /Karl)

Ligation of ADH2(Z),pirB, pdc, PpetE, adh2(Y) into pSB1A3 and self-ligation of the pSB1A3
Ligation with the pSB1A3 vector using the Quick Ligation Kit from NEB. Calculated amounts can be found here [[Media:Ligation Calc 090810.xls]]

Transformation of ADH2(Z),pirB, pdc, PpetE and pSB1A3-self-ligate into competent E. coli cells
Transformation was performed according to the dudes protocol. Apply 5µl of ligated mix on top of frozen competent E. coli cells. Let them thaw on ice. When completly thawn, stir gently with a pipette tip, heat shock at 42 °C for approximately 50 seconds. Back on ice and then at 1000µl of SOB medium.

Incubate for 60mins at 37°C and plate on selective LB plates (in our case LB + amp) over night at 37°C.

--Karl.brune 21:06, 10 August 2009 (UTC)