April 30th, 2010
I made a PCR to get confidence that the last restriction assay was correct. I am going to amplify the small region excised from the plasmid pSB1AK3; (the double terminator per se).
I am going to use the iGEM preffix and suffix primers that Nando gave me.
--- 3-5min (94°C) --- | ------ 30 - 45seg (60°C, 30 cycles) ------ 1min x kb (72°C) | ---- 3 - 5min (73°C) ---- (4°C)
The protocol I followed:
5μM Buffer PCR
2.5μM Primer Forward
2.5μM Primer Reverse
1μM DNA template (deluted)
1μM Taq Polymerase
H2O, Total: 50μM
I made a stock of the common reactants (6 samples).
Then I made a dilution of the DNA template, 4μM DNA in 20μM, from this dilution I took 1μM for the PCR. As a negative control I used a sample without DNA and 2 positive controls.
After the PCR I made an agarose gel to 1%.
The gel showed that the PCR didn't work; the positive controls did work which lead us to think that there wasn't enough template DNA.