IGEM:UNAM LCG/2009/Notebook/Hydrobium etli/2011/09/18

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pBBR1MCS-5 extraction by PCR

Abstract

I and Helena Reyes made a PCR from an extraction that she had made. The objective was to amplify and obtain the plasmid pBBR1MCS-5 without the RFP and its promoter in order to use the lasmid as vector to insert the anderson's promoter collection.

Apparently nothing was amplified, but it is curious that there are no bands neither for the lanes that had the supposed plasmid before the amplification, this means that probably the PCR did not fail, I think the problem was that we never introduced any template DNA (the plasmid) may be because the extraction failed.

Details

To make the PCR I used the NEB™ Phusion High-Fidelity DNA polymerase: details, to define the reactives consentrations and PCR steps I checked out the protocol for the enzyme: protocol.

I made 4 reactions, I had two different pBBR1MCS-5 samples, I made a 1/10 dilution for each. I ran a PCR for each sample diluted and not diluted.

Reactive ammounts

Reactive Ammount (μL)
MiliQ Water 24.5
Buffer 10
dNTPs 1c/u=4
PrimerF (suffix) 5
PrimerR (prefix) 5
Template DNA 1
Phusion polymerase 0.5
Total 50

Reactive details

  • Primers: I used the Suffix-forward and Prefix-reverse primers in order to amplify what was outside the preffix and suffix, which is the plasmid. The Tm for both plasmids were a little high: ~68°C.
  • Template DNA: I made a dilution 1 part per 10 of each plasmid before runing the PCR. I made the PCRs with both diluted and not diluted samples.

Run details

  • Hot start: 5 min, 95°C
  • Repetitions: 30 cycles
    • Denature: 10 sec, 98°C
    • Anneal: 30sec, 66°C
    • Extend: 1:00 min, 72°C
  • Final: 2 min, 72°C

Results

I ran a gel to check out the result of the PCR and the final concentration of the products. Nothing appear in the gel.

Gel details

  • Concentration: Agarose 1%
  • 2μL dye + 3μL sample
  • 2μL ladder 500 + 1μL dye.
  • Run: 35 min, 130 volts

Gel image

Lanes:

 1. pBBR1MCS-5 sample-3 no diluted (No PCR)
 2. pBBR1MCS-5 sample-3 no diluted (PCR, tube 1)
 3. pBBR1MCS-5 sample-3 1/10 diluted (PCR, tube 2)
 4. ladder
 5. pBBR1MCS-5 sample-4 no diluted (No PCR)
 6. pBBR1MCS-5 sample-4 no diluted (PCR, tube 3)
 7. pBBR1MCS-5 sample-4 no diluted (PCR, tube 4)
 8. empty


PBBR1MCS-5 PCR.JPG

Gel analysis

There was no ammplification, the lines in the gel are not visible. It is strange that lanes 1 and 5 are also empty because this means that there is no plasmid in there, so probably we never put any template DNA into the PCR. Maybe the plasmid extraction did not work out.

Notes

The tubes are in Helena's red rack, the PCR ones and the plasmid ones too.