Team:Groningen/Notebook/27 July 2009

From 2009.igem.org

Wet

GVP Cluster

Transporters

GlpF PCR 2 was add on gel again as noted below at accumulation.

PCR 1 was GlpF PCR1

A 2% agarose gel was used, a band of 105 bp was expected.

The band in row 2 was cut and purified using the zymo research Zymoclean TM Gel DNA Recovery Kit.

The purified PCR1 DNA and PCR2 DNA were used to run PCR3

GlpF PCR3

The results of the 1% agarose gel can be found at 28/07/09

Metal Accumulation

Gel with ArsR PCR((24/07/09)

ArsR/GlpF gel extraction

- Used zymo research Zymoclean TM Gel DNA Recovery Kit to extract ArsR, GlpF PCR2 and GlpF FullLenght from gel (cut from gel as shown in figure above).

GlpF PCR1

Vectors

• Restriction digest on pSB3K3-const. promoters and pSB1AC3-cons. promoters and pSB1A2-lac promoter
  • Plasmids isolated with sigma plasmid isolation kit from o/n culture 24-25 July
  • Restriction was done with 250-500ng plasmid

• • Expected fragments are
    • pSB1A2-Lac: 1400, 650, 180, 40bp
    • pSB3K3-const. promoter: 2620, 160bp
    • pSB3K3: 3100bp
    • pSB1AC3-const. promoter: 1770, 1319bp
    • pSB1AC3: 3400bp
  • Loaded on a 1% Agarose gel (TBE)
  • Contents are (from left to rigth):

1KB marker, pSB2K3-H, pSB2K3-M, pSB2K3-L, pSB2K3, pSB1AC3-H, pSB1AC3-M, pSB1AC3-L, pSB1AC3, pSB1A2-Lac1, pSB1A2-Lac2.

• • The fragments seem to be on the correct higth, though there are also extra bands seen, which may be uncut plasmid DNA.

• 'O/n cultures of pBAD
  • Pick 4 colonies of TOP10 + pSB1A2-pBAD/AraC
  • Grow o/n in ~4ml LB-amp in the warm room.

Dry

Finished RPU computations!