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Uppsala-Sweden/10 August 2009
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Purification of the digestion mix was performed with Nucleo Spin® Extract 2 PCR cleanup protocol from Clontech | Purification of the digestion mix was performed with Nucleo Spin® Extract 2 PCR cleanup protocol from Clontech | ||
Purified product (20µl) in 1.5ml eppendorfs. | Purified product (20µl) in 1.5ml eppendorfs. | ||
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| + | ==DNA Measurements== | ||
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| + | DNA concentration were measured for subsequent ligation. | ||
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| + | [[Media:afterpurification_090810.rtf]] | ||
==Ligation of ADH2(Z),pirB, pdc, PpetE, adh2(Y) into pSB1A3== | ==Ligation of ADH2(Z),pirB, pdc, PpetE, adh2(Y) into pSB1A3== | ||
Revision as of 21:12, 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 adjacent gel
Colony PCR was performed. 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 ([http://www.fermentas.com/fastdigest/index.html 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.rtf
Ligation of ADH2(Z),pirB, pdc, PpetE, adh2(Y) into pSB1A3
Ligation with the pSB1A3 vector using the Quick Ligation Kit from [http://www.neb.com/nebecomm/products/protocol2.asp NEB]. Calculated amounts can be found here Media:Ligation Calc 090810.xls
Transformation of ADH2(Z),pirB, pdc, PpetE, 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)
