Team:Groningen/Notebook/13 July 2009

From 2009.igem.org

(Difference between revisions)
(GVP Cluster)
(GVP Cluster)
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* 1μL  SpeI 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.
+
The cups were incubated in a heat block at 37°C for 15 min. followed by adding 4μL 6x loading buffer for gel electroforesis.
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25μL of each sample was loaded on a 1% agarose gel with EtBr and a 1kb ladder was used (see picture).
25μL of each sample was loaded on a 1% agarose gel with EtBr and a 1kb ladder was used (see picture).
-
[[]] [[Image:Generulers_1kb_marker_Fermentas.jpg]]
+
[[Image:Gel 2009-07-13 no.2.jpg|500px]] [[Image:Generulers_1kb_marker_Fermentas.jpg]]
-
:→ From left to right: Empty Slot, 1kb Marker,
+
:→ From left to right: 1kb Marker, Empty Slot, [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109], Empty Slot, [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100], Empty Slot, [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106], Empty Slot, GVP, Empty Slot
 +
 
 +
:→ The promotor parts (50bp) were not visible on gel after a run at 100V for 25 min. A second gel run for 10 min. at 100V showed the same result for promotor [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100], but had a vage outline of the correct fragment size (gel not shown). Purification of this part of the gel would yield a far too low concentration, and was therefor not attempted.
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:→ After the low concentration was determined, the cup was disposed of because a higher concentration is required!
:→ After the low concentration was determined, the cup was disposed of because a higher concentration is required!
 +
 +
 +
'''Inoculation of TY-medium'''
 +
 +
TY-medium (5mL) was inoculated with a tip of glycerol stocks:
 +
 +
* GVP no.1
 +
* [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109]
 +
* [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100]
 +
* [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106]
 +
 +
and 5μL ampicilin (1000x). The tubes were placed in a 37°C shacker for over night incubation at 200 rpm.
===Transporters===
===Transporters===

Revision as of 14:51, 13 July 2009

Igemhomelogo.png

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 electroforesis.


Gel electroforesis

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

Gel 2009-07-13 no.2.jpg Generulers 1kb marker Fermentas.jpg

→ From left to right: 1kb Marker, Empty Slot, BBa_J23109, Empty Slot, BBa_J23100, Empty Slot, BBa_J23106, Empty Slot, GVP, Empty Slot
→ The promotor parts (50bp) were not visible on gel after a run at 100V for 25 min. A second gel run for 10 min. at 100V showed the same result for promotor BBa_J23100, but had a vage outline of the correct fragment size (gel not shown). Purification of this part of the gel would yield a far too low concentration, and was therefor not attempted.


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!


Inoculation of TY-medium

TY-medium (5mL) was inoculated with a tip of glycerol stocks:

and 5μL ampicilin (1000x). The tubes were placed in a 37°C shacker for over night incubation at 200 rpm.

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