Team:Groningen/Notebook/13 July 2009

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Wet

GVP Cluster

Restriction for Assembly

The vector containing GVP cluster was cut with EcoRI and XbaI to create correct ends.

  • 6μL MQ
  • 10μL plasmid in MQ
  • 2μL Fast digest buffer
  • 1μL EcoRI fast digest enzyme
  • 1μL XbaI fast digest enzyme

The vectors containing promotors BBa_J23109, BBa_J23100 and BBa_J23106 were cut with EcoRI and SpeI.

  • 0μL MQ
  • 16μL plasmid in MQ (BBa_J23109 and BBa_J23106)
  • 2μL Fast digest buffer
  • 1μL EcoRI fast digest enzyme
  • 1μL SpeI fast digest enzyme

-

  • 6μL MQ
  • 10μL plasmid in MQ (BBa_J23100)
  • 2μL Fast digest buffer
  • 1μL EcoRI fast digest enzyme
  • 1μL SpeI fast digest enzyme

The cups were incubated in a heat block at 37°C for 15 min. followed by adding 4μL 6x loading buffer for gel electroforese.


Gel electroforesis

25μL of each sample was loaded on a 1% agarose gel with EtBr and a 1kb ladder was used (see picture).

[[]] Generulers 1kb marker Fermentas.jpg

→ From left to right: Empty Slot, 1kb Marker,


Gel Purification of GVP

A standard kit for PCR-product purification was used for gel purification

  • 120mg of gel containing the desired fragment was dissolved in 500μL binding buffer by heating to 60°C for 10 min.
  • the column was prepared with 500μL Preparation Solution and centrifuged for 1 min.
  • dissolved gel was transfered to the column and centrifuged for 1 min.
  • column was washed with 750μL Wash Solution and centrifuged twice 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-vector eluted in MQ

  • 4.3 ng/μL
  • 1.42 (260/280)
  • 0.60 (260/230)
→ After the low concentration was determined, the cup was disposed of because a higher concentration is required!

Transporters

PCR program Temperature Time
Denaturing 95° 2.00 min
Start Cycles 25X
Denaturing 95° 30 sec
Annealing 55° 20 sec
Elongation 72° 4.10 min
End cycles
Final elongation 72° 10 min
Hold Forever

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