IGEM:UNAM LCG/2009/Notebook/Hydrobium etli/2011/09/03

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Promoters PCR amplification

Abstract

I made a PCR to amplify and extract the promoters: J23101, J23102, J23103, J23104, J23107, J23108, J23109, J23110, J23113, J23114, J23116.

It all went well, now I have all the Anderson Collection in a construction flanked by the standar preffix and suffix. The construction includes a RBS an RFP and a doble terminator at the end (aprox 1 kb in total).

The next step is to digest the constructions with EcoRI and PstI in order to ligate them with the plasmid pBBR1MCS5 backbone.

Details

To make the PCR 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 11 reactions (one for each promoter to amplify), the next table shows the reactives ammounts I used for a single reaction.

Reactive ammounts

Reactive Ammount (μL)
MiliQ Water 33.5
Buffer 5
dNTPs 1c/u=4
MgCl2 1.5
PrimerF 2
PrimerR 2
Template DNA 1.5
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 between the preffix and suffix.
  • Template DNA: I made a dilution 1 part per 20 of each plasmids before runing the PCR.

Run details

  • Hot start: 2 min, 94°C
  • Repetitions: 30 cycles
    • Denature: 30 sec, 94°C
    • Anneal: 30sec, 55°C
    • Extend: 1:05 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.

Gel details

  • Concentration: Agarose 1%
  • 2μL dye + 3μL sample
  • 5μL ladder
  • Run: 38 min, 120 volts

Gel image

Lanes:

 1. J23101
 2. J23102
 3. J23103
 4. J23104
 5. J23107
 6. J23108
 7. J23109
 8. J23110
 9. J23113
 10. J23114
 11. J23116
 12. Ladder //cuál ladder?
 The remaining lanes are not of interest for this analysis

Image:PCR_amplif_11_promoters.JPG

Gel analysis

By the lines we can say that all the parts were succesfully ammplified, they are all near 1kb which is the expected size and they are all near a quantity of 120ng (the dark lines un the ladder correpond to 60ng) except for J23104 (4th line) which appears to be 50ng aprox.


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