User:Jose Fabricio Lopez/Notebook/Logbook/2011/08/18

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Objectives

  • PCR of B0015.
  • PCR of pSB1T3.
  • See the in Gel.
  • Restriction of B0015 and pSB1T3 backbone

Working

PCR of B0015

The point of this PCR is get double terminator and insert it into pSB1T3 backbone.

  • Amplified with preffix and suffix primers.


Reagents μL
Buffer Taq Standard 10X 5
dNTPs 8mM 1.25
Oligo F/R 2.5/2.5
DNA B0015 1
H2O 36.75
Taq DNA 1
Total 50μL

Gel B0015

  • Lane 1. Ladder
  • Lane 2 & 3. B0015

It's dirty but usefull

PCR of pSB1T3

  • PCR of pSB1T3/RFP, to amplify just backbone.
Reagents μL
Buffer Taq Platinum 10X 5
dNTPs 8mM 5
MgCl 1.5
Oligo SuffixF/PrefixR 2/2
DNA pSB1T3 1
H2O 33
Taq Platinum DNA Pol .5
Total 50μL

All 2X. Two PCRs.

Gel PCR of pSB1T3 backbone

  • Lane 1 Ladder
  • Lane 2 & 3 Backbone pSB1T3

As expected :) Both amplified.

Purification of PCR product

Both were purified with Roche´s Kit for PCR purification

Restriction

I performed restriction with EcoRI and PstI.

Poner esto RECUERDA!!!




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