Team:Groningen/Notebook/31 August 2009

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Wet

GVP Cluster

Concentrations

Plasmid Conc. ng/μL 260/280 260/230 -20 box (michael Restriction Control
pSB1A2-pLacI-GVP 41.2 1.85 2.18 ? Yes (EcoRI/PstI)
pSB1A2-pBad/araC-GVP 421.7 1.84 2.34 ? Yes (EcoRI/PstI)
pSB1AC3-H 2.43.1 1.86 2.17 All Used Yes (Glycerol Stock)


Restriction for Assembly

The vector pSB1A2 containing the pLacI/GVP and pBad/araC/GVP composite parts were cut with PstI and EcoRI to create correct ends for insert into pSB1AC3, which was also cut with EcoRI and PstI.

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 15.0 x 3.0 1.0 x x 1.0
pSB1A2-pBad/araC-GVP 4.0 12.0 3.0 1.0 x x 1.0
pSB1AC3-High 6.0 9.0 3.0 1.0 x x 1.0

Restriction was kept at 37C for 30 min. and put on ice until used for gel purification.

Purification

240px Generulers 1kb marker Fermentas.jpg

→ From left to right: 1kB ladder, pSB1A2-pLacI-GVP (2x) ,Empty Slot, pSB1A2-pBAD-AraC-GVP (2x) , Empty Slot, pSB1AC3-H (2x)


A "Agarose Gel DNA Extraction Kit" Standard Protocol from Roche Applied Science.

→ In step ten, 30μL MQ was added to the dry pellet and incubated at room temperature.


Concentrations

Plasmid Conc. ng/μL 260/280 260/230 -20 box (michael Restriction Control
pLacI-GVP (EcoRI/PstI) 5.0 1.47 1.04 ? Yes (EcoRI/PstI)
pBad/araC-GVP (EcoRI/PstI) 24.0 1.76 1.53 ? Yes (EcoRI/PstI)
pSB1AC3 (EcoRI/PstI) 24.4 1.77 1.69 ? Yes (EcoRI/PstI)

Ligation

A total amount of vector of 125-200ng was used (promoter-GVP) in a 1:2 and 1:3 ratio with insert.

(1:3)

  • 3 uL Ligase buffer
  • 1 ul T4 Ligase
  • 8 uL plasmid pSB1AC3 digested with EcoRI and PstI
  • 8 uL insert pBad-araC-GVP restricted with EcoRI and PstI

(1:2)

  • 3 uL Ligase buffer
  • 1 ul T4 Ligase
  • 11 uL plasmid pSB1AC3 digested with EcoRI and PstI
  • 5 uL insert pLac-GVP restricted with EcoRI and PstI

Incubate:

  • 25°C 50min.
  • kept on ice for 10min.

Tranformation

  • add 10uL of the ligation product to 50uL competent E.coli TOP10 cells.

Incubate:

  • 30 min @ ice
  • 90 sec 42°C
  • 2 min @ ice
  • add 800uL LB-medium
  • incubate for 1 h at 37°C
  • plate on LB-amp100 plates


→ Positive control was BBa_J61002-J23101 plasmid, and negative control was MQ.

Transporters

HmtA

The cloning strategy will be changed. First we will make a PCR2 and a PCR1.2 where F2-mut1rc plus Rev-mut2rc will generate a HmtA where we will add a prefix via a PCR with F1-REV. And than ready for cloning. But first we wait for a kit.

An aditional PCR started to get more megaprimer.

Metal Accumulation

Vectors

Dry

April
MTWTFSS
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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
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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
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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
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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
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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
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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