"

From 2012.igem.org

Contents 1 6th August 2 7th August 3 8th August 4 9th August 5 10th August

6th August

• Sending of the B1 sample to sequencing with two pairs of primers
  • K-274100 Forward and Reverse
  • Plasmid pSB1A2 Forward and Reverse

7th August

• Liquid culture of B1 in order to prepare a glycerol stock
  • Receipt of new primers
  • 2 Forward
  • 3 Reverse
  • Plasmid Reverse

8th August

Amplification of the plasmid pSB1A2, BBa_K274100 and BBa_K115017 by PCR with the new primers. Visualization by electrophoresis on a 2% Agarose gel for BBa_K115017 and a 0.8% Agarose gel for BBa_K274100 and the plasmid pSB1A2. We are expecting a band at 147 bp for BBa_K115017, 3408 bp for BBa_K274100 and 2079 for the plasmid.

PCR program used:

9th August

New amplification by PCR of the plasmid pSB1A2, BBa_K274100 and BBa_K115017 as it has been done the day before.

• Digestion by EcoRI of the plasmid that contains BBa_K274100.

10th August

Amplification by PCR of BBa_K274100 already digested by EcoRI. Visualization by electrophoresis on a 0.8% Agarose gel.

PCR program used:

Miniprep of BBa_K274100 followed by Nanovue to determine the concentration of the sample
Miniprep of the Plasmid pSB1A2 followed by Nanovue to determine the concentration of the sample

• Digestion by HindIII of the plasmid pSB1A2 in order to linearize it.

Digestion of BBa_K115017 by DPNI to eliminate the plasmid matrix. Visualization by electrophoresis on a 2% Agarose gel. We are expecting a band at 123 bp.