Team:Groningen/Notebook/5 August 2009

From 2009.igem.org

(Difference between revisions)
(GVP Cluster)
m (GVP Cluster)
Line 11: Line 11:
:→ {{todo}} buy a nice cake and eat it ;)
:→ {{todo}} buy a nice cake and eat it ;)
:→ {{todo}} isolate plasmids from overnight precultures
:→ {{todo}} isolate plasmids from overnight precultures
-
:→ {{todo}} perform a ligation between cut vector and GVP fragment
+
:→ {{done}} perform a ligation between cut vector and GVP fragment
:→ {{todo}} transform E.coli TOP10 competent cells with ligation product
:→ {{todo}} transform E.coli TOP10 competent cells with ligation product
'''Ligation'''
'''Ligation'''
-
:*2 uL Ligase buffer
+
:* 2 uL Ligase buffer
-
:*1 ul T4 Ligase
+
:* 1 ul T4 Ligase
-
:*20 uL plasmid pSB1AC3 digested with PstI and SpeI
+
:* 8 uL plasmid pSB1AC3 digested with PstI and SpeI
-
:*6 uL insert GVP restricted with XbaI and PstI
+
:* 4 uL insert GVP restricted with XbaI and PstI
''Incubate:''
''Incubate:''
-
:*16°C 60min.
+
:* 25°C 30min.
-
:*65°C 10min.
+
:* kept on ice for 10min.
'''Tranformation'''
'''Tranformation'''
-
:* add 15 uL of the pSB1AC3-GVP ligation product to 50uL competent E.coli TOP10 cells.
+
:* add 10uL of the pSB1AC3-GVP ligation product to 50uL competent E.coli TOP10 cells.
''Incubate:''
''Incubate:''
:* 30 min @ ice
:* 30 min @ ice
-
:* 5 min 37°C
+
:* 50 sec 42°C
-
:* 5min @ ice
+
:* 2 min @ ice
:* add 800uL LB-medium
:* add 800uL LB-medium
-
:* incubate for 1 h at 37°
+
:* incubate for 1 h at 37°C
-
:* plate on LB-AMP plates
+
:* plate on LB-amp<sub>50</sub> plates
==Dry==
==Dry==
{{Team:Groningen/Notebook/Day/Footer}}
{{Team:Groningen/Notebook/Day/Footer}}

Revision as of 10:01, 5 August 2009

Igemhomelogo.png

It's my birthday!!! (Michael)

So cake it is....

Wet

GVP Cluster

TODO buy a nice cake and eat it ;)
TODO isolate plasmids from overnight precultures
DONE perform a ligation between cut vector and GVP fragment
TODO transform E.coli TOP10 competent cells with ligation product

Ligation

  • 2 uL Ligase buffer
  • 1 ul T4 Ligase
  • 8 uL plasmid pSB1AC3 digested with PstI and SpeI
  • 4 uL insert GVP restricted with XbaI and PstI

Incubate:

  • 25°C 30min.
  • kept on ice for 10min.

Tranformation

  • add 10uL of the pSB1AC3-GVP ligation product to 50uL competent E.coli TOP10 cells.

Incubate:

  • 30 min @ ice
  • 50 sec 42°C
  • 2 min @ ice
  • add 800uL LB-medium
  • incubate for 1 h at 37°C
  • plate on LB-amp50 plates

Dry

April
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May
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June
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29 30
July
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August
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31
September
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October
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November
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