User:Nelson Augusto Berrocal/Notebook/WiFi Coli 2010 Wet Lab/2010/06/22

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June 22nd, 2010
Today I made a PCR in order to amplify LuxAB from Vibrio fischeri MJ11's genomic DNA, I made a total of four reactions

1. The first two reactions were made with Rtth DNA Polymerase in order to amplify LuxAB from VF MJ11's genomic DNA following the next protocol:

1st solution

2μL template DNA (Genomic DNA)

6μL Bufer 3.3x

3μL Mg(Ac)2

2.5μL dNTPs

2.5μL Forward Primer

2.5μL Reverse Primer

11.5μL H2O

30μL TOTAL

After a hotstart I added the 2nd solution

10.5μL H2O

9μL Buffer 3.3x

0.5μL Rtth polymerase

TOTAL 20μL

2.The next two reactions were made with Platinum Taq Polymerase of Invitrogen; the third one amplified LuxAB and the fourth one amplified a control of ~2.5kb, following the next protocol:

5μL 10xPCR Buffer minus M 1μL 10mM dNTP mixture

1.5μL 50mM MgCl2

1μL Primer mix(10μM each)

2μL Template DNA

0.2μL Platimum Taq Polymerase

H2O

Total:50μL

The PCR parameters were like this:

94°C-5min---STOP--- | 94°C-30s-55°C-30°C-72°C-3:30min(35 cycles)--- | 72°C-5min---4°C>

Once the PCR was done I made an agarose gel to 0.8% to regard if the PCR worked, and I obtained the following results:

Reaction1 Rtth Didn't ampliffied --- Reaction2 Rtth ampliffied! --- Reaction3 Taq platinum didn't ampliffied --- Control ampliffied


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