User:Nelson Augusto Berrocal/Notebook/WiFi Coli 2010 Wet Lab/2010/04/30

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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.

PCR

--- 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 MgCl2

2.5μM DNTPs

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.


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