Team:Groningen/Notebook/13 August 2009

From 2009.igem.org

(Difference between revisions)
(New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== :→ {{todo}} make fresh LB-agar medium :→ {{todo}} inocculate o.n. cultures for glycerol stocks :→ {{todo}} ligatio...)
(GVP Cluster)
Line 7: Line 7:
:→ {{todo}} make fresh LB-agar medium
:→ {{todo}} make fresh LB-agar medium
:→ {{todo}} inocculate o.n. cultures for glycerol stocks
:→ {{todo}} inocculate o.n. cultures for glycerol stocks
-
:→ {{todo}} ligation of GVP into vectors
+
:→ {{done}} ligation of GVP into vectors
:→ {{todo}} transformation of E.coli TOP10 with ligation product
:→ {{todo}} transformation of E.coli TOP10 with ligation product
 +
 +
'''Ligation'''
 +
 +
(1:3)
 +
:* 4 uL Ligase buffer
 +
:* 1 ul T4 Ligase
 +
:* 9 uL plasmid pSB1AC3-H digested with PstI and SpeI
 +
:* 9 uL insert GVP restricted with XbaI and PstI
 +
 +
(1:3)(2x)
 +
:* 4 uL Ligase buffer
 +
:* 1 ul T4 Ligase
 +
:* 12 uL plasmid pSB3K3-H/L digested with PstI and SpeI
 +
:* 5 uL insert GVP restricted with XbaI and PstI
 +
 +
''Incubate:''
 +
:* 25°C 60min.
 +
:* kept on ice for 10min.
 +
 +
'''Tranformation'''
 +
:* add 10uL of the pSB1AC3-H-GVP and pSB3K3-H/L-GVP ligation products 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
 +
:* concentrate in 100uL LB-medium
 +
:* plate on LB-amp<sub>50</sub> plates
===Transporters===
===Transporters===

Revision as of 08:42, 13 August 2009

Igemhomelogo.png

Wet

GVP Cluster

TODO make fresh LB-agar medium
TODO inocculate o.n. cultures for glycerol stocks
DONE ligation of GVP into vectors
TODO transformation of E.coli TOP10 with ligation product

Ligation

(1:3)

  • 4 uL Ligase buffer
  • 1 ul T4 Ligase
  • 9 uL plasmid pSB1AC3-H digested with PstI and SpeI
  • 9 uL insert GVP restricted with XbaI and PstI

(1:3)(2x)

  • 4 uL Ligase buffer
  • 1 ul T4 Ligase
  • 12 uL plasmid pSB3K3-H/L digested with PstI and SpeI
  • 5 uL insert GVP restricted with XbaI and PstI

Incubate:

  • 25°C 60min.
  • kept on ice for 10min.

Tranformation

  • add 10uL of the pSB1AC3-H-GVP and pSB3K3-H/L-GVP ligation products 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
  • concentrate in 100uL LB-medium
  • plate on LB-amp50 plates

Transporters

Metal Accumulation

Vectors

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