Team:UAB-Barcelona/PCRN

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

Annealing temperature

nirk (270 pb): as lowest temperature is 60ºC, the temperature we should set in the thermociclator would be 55ºC. But it didn't work properly at this temperature, so we set the temperature to 52ºC and 49ºC. PCR in both cases worked properly http://2009.igem.org/wiki/images/1/12/NirK.jpg

Marker, 3x49ºC, controls, marker, 3x52ºC, controls

From left side to right: lambda-DNA hinIII; 3 x nirk 49ºC; control without primers; control without DNA template; lambda-DNA hinIII; 3 x nirk 52ºC; control without primers; control without DNA template; lambda-DNA hinIII

Annealing temperature

nsr(500 pb): the temperature is 76ºC, so we made the PCR at 71ºC. It didn't work. But when we tried with 65 and 68ºC it worked properly.

http://2009.igem.org/wiki/images/f/f3/Pnsr.jpg lambda-DNA hinIII; 4 x nsr 65ºC; control without DNA template; control without primers; lambda-DNA hinIII; 4 x nsr 68ºC; control without DNA template; control without primers; lambda-DNA hinIII

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

http://2009.igem.org/wiki/images/b/b1/Purpnir.jpg

Agarose gel nirk purification

From left side to right: lambda-DNA hinIII; 3 x nirk 52ºC; control without primers; 2x nirk 49ºC; lambda-DNA hinIII

http://2009.igem.org/wiki/images/1/14/Purnsr.jpg

Agarose gel nsr purification

From left side to right: lambda-DNA hinIII; 3 x nsr 65ºC; 3 x nsr 68ºC; lambda-DNA hinIII