Team:Groningen/Notebook/7 August 2009

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Wet

GVP Cluster

TODO isolate plasmids from overnight precultures
TODO perform a restriction on isolated plasmid (should result in 6000 and 3000bp fragments)
TODO plate on cultures for pure colony over the weekend
TODO cut pSB3K3 plasmid and GVP containing plasmid for ligation
TODO purify wanted fragments
www.sigmaaldrich.com

Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids pSB1AC3 with high, medium and low constitutive promoters and GVP with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".

  • From each tube 4mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
  • Plasmids were eluted with 20μL MQ and stored in the fridge

Concentration of Plasmids

Plasmid ng/μL 260/280 260/230 Control
pSB3K3-H 25.0 ? ? ?
pSB3K3-M 32.6 ? ? ?
pSB3K3-L 33.9 ? ? ?
GVP (biobrick) 484.0 ? ? ?
pSB1AC3-H-GVP-amp 106.6 2.00 2.44 ?
pSB1AC3-M-GVP-amp 165.2 1.92 2.40 ?
pSB1AC3-L-GVP-amp 155.0 1.95 2.44 ?
pSB1AC3-H-GVP-amp-conc. 116.3 1.96 2.43 ?
pSB1AC3-M-GVP-amp-conc. 108.7 1.95 2.46 ?
pSB1AC3-L-GVP-amp-conc. 302.8 1.88 2.45 ?
pSB1AC3-H-GVP-amp/chl-conc. 257.1 1.92 2.41 ?
pSB1AC3-M-GVP-amp/chl-conc. 224.7 1.94 2.37 ?
pSB1AC3-L-GVP-amp/chl-conc. 166.1 1.95 2.44 ?
pSB1AC3-L-GVP-amp/chl-oud 223.9 1.91 2.31 ?

Transporters

Metal Accumulation

  • 2nd PCR to amplify SmtA and GST-SmtA
    • Use pGEX-3X-GST-SmtA to amplify SmtA-GST and SmtA
    • Use pET29a-SmtA to amplify SmtA
    • Program: MT NIENKE (Left PCR machine)
      • Globally: 10cycles touchdown from 65-50°C, 25 cycles with Tm gradient from 60-50°C (4steps), elongation time of 1:20.
    • Mix
MM NH4 21uL
Primer fw [10mM] 1uL
Primer rev [10mM] 1uL
Vector 1uL
HomeTaq pol 1uL
    • Run program o/n
    • Check on gel
  • Transform E. coli with pGB68 (mymT)
    • Use pGB68 from filter resuspended in 40uL MQ.
    • As neg control use pSB1AC3 (incl ccdB), as pos control use pSB1AC3-low promoter.
    • Mix 2-4uL of vector with 50uL chemically competent cells (prepared 31 aug)
    • Transform using heat-shock @ 37°C

Vectors

Dry

April
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
May
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
November
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30