Team:UAB-Barcelona/PCRP

PCR of phosphate detection sequences
We used GoTaq Green Master Mix (Promega) 12

Annealing temperature

phoA (100 pb): as annealing temperature is 74ºC, the temperature we should set in the thermociclator would be 69ºC. But it didn't work properly at this temperature, so we set the temperature to 66ºC and 64ºC. PCR in both cases worked properly

http://2009.igem.org/wiki/images/3/37/PhoA.jpg

lambda-DNA hinIII; 3 x phoa 64ºC; control without primers; control without DNA template; lambda-DNA hinIII; 3 x phoa 66ºC; control without primers; control without DNA template; lambda-DNA hinIII

Purification of phoA
We purified the bands with the kit Wizard SV Gel and PCR Clean-Up System (PROTOCOL).

http://2009.igem.org/wiki/images/1/1d/Purphoa.jpg

Agarose gel phoa purification

From left side to right: lambda-DNA hinIII; 3 x phoa 64ºC; control without primers; control without DNA template; lambda-DNA hinIII; 3 x phoa 66ºC; control without primers; control without DNA template; lambda-DNA hinIII