Team:Groningen/Notebook/15 July 2009

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GVP Cluster

Restriction for Assembly

The vector (BBa_J61035) containing GVP cluster was cut with PstI and XbaI to create correct ends for insert in promotor vector (BBa_J61002).

  • 10μL MQ
  • 6μL plasmid in MQ (2μg) (329.7 ng/μL)
  • 2μL Fast digest buffer
  • 1μL PstI fast digest enzyme
  • 1μL XbaI fast digest enzyme

The vector (BBa_J61002) containing promotor BBa_J23100 was cut with SpeI and PstI.

  • 4μL MQ
  • 12μL plasmid in MQ (155.4 ng/μL)
  • 2μL Fast digest buffer
  • 1μL SpeI fast digest enzyme
  • 1μL PstI fast digest enzyme

The cups were incubated in a heat block at 37°C for 30 min. followed by adding 4μL 6x loading buffer for gel electroforesis.


Gel electroforesis

24μL of each sample was loaded on a 1% agarose gel (devided over two slots) with EtBr and a 1kb ladder was used (see picture).

Gel 2009-07-15.jpg Generulers 1kb marker Fermentas.jpg

→ From left to right: Empty slot, 1kb Marker, Empty slot, HmtA PCR reaction, Empty slot, (2x) GVP, Empty slot, (2x) BBa_J23100, and Empty slot


Gel Purification of GVP

"High Pure PCR Product Purification Kit" from Roche was used for gel purification.

  • ~200mg of gel containing the desired fragment was dissolved in 500μL binding buffer by heating to 60°C for 10 min. and carefully vortexing.
  • 250μL isopropanol was added to the tube and vortexed
  • dissolved gel was transfered to the column and centrifuged for 1 min.
  • column was washed with 500μL Wash Solution, centrifuged for 1 min., 200μL Wash solution was added and centrifuged for 1 min.
  • 15μL MQ was applied to the column, incubated for 3 min. at room temperature and centrifuged for 1 min. at full speed.


Concentration of GVP-vector

The concentration of isolated cut vector of GVP with EcoRI and XbaI was determined with the use of a nano-drop.

GVP-cluster eluted in MQ

  • 33.5 ng/μL
  • 1.65 (260/280)
  • 0.77 (260/230)

BBa_J23100 + vector eluted in MQ

  • 14.2 ng/μL
  • 1.78 (260/280)
  • 1.01 (260/230)
→ Concentrations are just high enough for a ligation reaction to be performed, some additional tuning of the procedure is required!!


Ligation of GVP into BBa_J23100 vector

Mix:

  • 1 μL T4 Ligase buffer
  • 1 μL Vector (J23100 restriction fragment, 14.2 ng/μL)
  • 7.6 μL Insert (GVP gene cluster, 33.5 ng/μL)
  • 0.5 μL T4 Ligase

Method:

  • The mixture is incubated 30 min, 26°C
  • To inactivate Ligase 10 min 70°C


Transformation of E.coli TOP10 cells with GVP + J23100

  • 2 μL Ligation mix added to cells from -80°C glycerol stock
  • 30 min incubation on ice
  • 5 min heat shock 37°C
  • 5 min on ice
  • Cells added to 3 ml Ty in a reaction tube
  • Incubation 1 h in waterbath shaker 37°C


Transporters

We decided to make MasterMixes of our own. All but template, primers and taq.

1X MasterMix Buffer NH SO
Component
dNTP 2.0 mM 8 uL
MgCl 2.0 mM 80 uL
taq buffer (NH)SO 1X 100 uL
MQ 652 uL


1X MasterMix Buffer KCl
Component
dNTP 2.0 mM 8 uL
MgCl 1.5 mM 60 uL
taq buffer (NH)SO 1X 100 uL
MQ 672 uL


MasterMix3
Component Mg/K
MQ 28.5 uL
F mut1 2 uL
R mut2 2 uL
dNTP 2 uL
MgCl 3 uL
taq buffer KCl 5 uL
taq buffer (NH)SO 5 uL
taq polymerase 2.5 uL
PCR 3 program Temperature Time
Denaturing 95° 2.00 min
Start Cycles 25X
Denaturing 95° 30 sec
Annealing 55° 20 sec
Elongation 72° 2.10 min
End cycles
Final elongation 72° 10 min
Hold Forever

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
          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
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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