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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