Team:Groningen/Notebook/4 September 2009

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Wet

GVP Cluster

BBa_F2620


O.n. precultures

→ All four o.n. precultures showed bacterial growth, and could be used to isolate plasmid. The four are registry vector pSB1A2 with pLacI/pBad-araC and GVP. The isolated plasmid is used for transformation into pSB2K3 and sent for sequencing.


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Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing the above mentioned plasmids 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 30μL MQ and stored in the fridge


Concentrations

Plasmid Conc. ng/μL 260/280 260/230 -20 box (michael Restriction Control
pSB1A2 pLacI-GVP no.1 405.3 1.85 2.36 D-6 Yes (EcoRI/PstI)
pSB1A2 pLacI-GVP no.2 34.0 2.01 2.20 D-7 Yes (EcoRI/PstI)
pSB1A2 pBad/araC-GVP no.1 27.5 2.09 2.16 D-8 Yes (EcoRI/PstI)
pSB1A2 pBad/araC-GVP no.2 33.3 1.96 2.06 D-9 Yes (EcoRI/PstI)


Restriction for Assembly

The vector pSB1A2 containing the pLacI and pBad-araC with GVP composite parts were cut with PstI and EcoRI to create correct ends for insert into pSB2K3, which was also cut with EcoRI and PstI (4x) on 3-9.

Plasmid Amount μL MQ μL Fast digest buffer EcoRI fast digest enzyme XbaI fast digest enzyme SpeI fast digest enzyme PstI fast digest enzyme
pSB1A2 pLacI-GVP no.1 3.0 13.0 3.0 1.0 x x 1.0
pSB1A2 pLacI-GVP no.2 16.0 x 3.0 1.0 x x 1.0
pSB1A2 pBad/araC-GVP no.1 16.0 x 3.0 1.0 x x 1.0
pSB1A2 pBad/araC-GVP no.2 16.0 x 3.0 1.0 x x 1.0

Restriction was kept at 37C for 40 min. and put on ice until used for gel purification.


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Gel Purification

  • In step 7 an amount of 10μL MQ was added to elute the DNA fragments.

Transporters

Metal Accumulation

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