IGEM:UNAM LCG/2009/Notebook/Hydrobium etli/2011/09/20

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pBBR1MCS backbone PCR extraction

Abstract

I made a PCR to extract the backbone of pBBR1MCS. The plasmid is standarized so I used the prefix fwd and suffix reverse primers. I need this backbones to insert the entire Anderson collection in R. etli. I used the plasmids extracted by Vladimir (see the previous entrance). I ran a gel (on 21/09/2011) to see if the PCRs were well, but unfourtunatly nothing appeared.

Details

I ran PCRs from the 4 plasmid extractions made by Vladimir to extract the backbone. I used the Invitrogen's™ Platinum® Taq DNA polymerase, to define the reactives consentrations and PCR steps I checked out the manual online for the enzyme: PCR taq platinum manual.

I made 4 reactions, one for eachplasmid extraction made by Vladimir: pBBR1MCS 1, pBBR1MCS 2, pBBR1MCS DH5α and pBBR1MCS S17.

Reactive ammounts

Reactive Ammount (μL)
MiliQ Water 39.5
Buffer 5
dNTPs 0.4mM 1
MgCl2 1.5
PrimerF prefix 1
PrimerR suffix 1
Template DNA 0.5
Taq plat polymerase 0.5
Total 50

Reactive details

  • Primers: I used the Prefix-forward and Suffix-reverse primers in order to amplify what was ouside the preffix and suffix, the backbone.
  • I did not dilute the template, I just added too little. I also added a little bit more of primers and dNTPs to the stock. I added less template DNA to the S17 reaction because it was more concentrated than the other plasmids.

Run details

  • Hot start: 4 min, 94°C
  • Repetitions: 30 cycles
    • Denature: 30 sec, 94°C
    • Anneal: 30sec, 57°C
    • Extend: 5:00 min, 72°C
  • Final: 9 min, 72°C


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