Team:UAB-Barcelona/PCRP

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Latest revision as of 02:39, 22 October 2009

Documento sin título

PCR of phosphate detection sequences

We used GoTaq Green Master Mix (Promega)1 2

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

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).

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

@@ Line 1: / Line 1: @@
 {{Template:UAB-Barcelona2}}
-==PCR of nitrite detection sequences==
+==PCR of phosphate detection sequences==
 We used GoTaq Green Master Mix (Promega)<html><a href="https://static.igem.org/mediawiki/2009/e/ea/Gotaq1.jpg">1</a><a href="https://static.igem.org/mediawiki/2009/e/ea/Gotaq2.jpg">2</a></html>
@@ Line 7: / Line 7: @@
 '''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
+''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
-https://static.igem.org/mediawiki/2009/1/12/NirK.jpg
-Marker, 3x49ºC, controls, marker, 3x52ºC, controls
+https://static.igem.org/mediawiki/2009/3/37/PhoA.jpg
-'''Annealing temperature'''
+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 <html><a href="https://static.igem.org/mediawiki/2009/1/1e/Protocols.pdf">(PROTOCOL)</a></html>.
+https://static.igem.org/mediawiki/2009/1/1d/Purphoa.jpg
+'''Agarose gel phoa purification'''
-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.
-https://static.igem.org/mediawiki/2009/f/f3/Pnsr.jpg
+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
-Marker, 4x65ºC, controls, marker, 4x68ºC, controls