Team:Groningen/Notebook/31 July 2009

From 2009.igem.org

(Difference between revisions)
(Transporters)
(Transporters)
Line 9: Line 9:
*Restiction/ligase and transformation of GlpF in psB1AC3
*Restiction/ligase and transformation of GlpF in psB1AC3
-
:* '''Restiction'''
+
: '''Restiction'''
{|
{|
|10ul
|10ul
Line 33: Line 33:
-
:*'''Ligation'''
+
:'''Ligation'''
{|
{|
|2 uL
|2 uL
Line 53: Line 53:
|}
|}
-
*'''Tranformation'''
+
:'''Tranformation'''
-
:* add 4 uL of the GlpFpsB1AC3 ligation mixture to 50uL competent e.coli top10 cells.
+
::* add 4 uL of the GlpFpsB1AC3 ligation mixture to 50uL competent e.coli top10 cells.
Incubate:
Incubate:
-
::*30 min @ ice
+
:::*30 min @ ice
-
::*5 min 37°
+
:::*5 min 37°
-
::*5min @ ice
+
:::*5min @ ice
-
:*add 800uL LB
+
::*add 800uL LB
-
::* incubate for 1 h at 37°
+
:::* incubate for 1 h at 37°
-
:* plate on LB-AMP plates
+
::* plate on LB-AMP plates
===Metal Accumulation===
===Metal Accumulation===

Revision as of 12:38, 31 July 2009

Igemhomelogo.png

Wet

GVP Cluster

Transporters

GlpF

  • Restiction/ligase and transformation of GlpF in psB1AC3
Restiction
10ul GlpF PCR
2 ul Buffer
05.ul EcoRI
0.5 ul SpeI
7 uL MQ
  • 37 30min
  • Inactivation on Gel
  • Extraction from gel


Ligation
2 uL Ligase buffer 16° 1h → 65° 10 min
1 ul T4 Ligase
11 uL GlpF digested with EcoRI and SpeI 42° 2 min →
11 uL psB1AC3 digested with EcoRi and SpeI
Tranformation
  • add 4 uL of the GlpFpsB1AC3 ligation mixture to 50uL competent e.coli top10 cells.

Incubate:

  • 30 min @ ice
  • 5 min 37°
  • 5min @ ice
  • add 800uL LB
  • incubate for 1 h at 37°
  • plate on LB-AMP plates

Metal Accumulation

ArsR-MBP fusion

  • colony PCR MBP:
colony PCR MBP
Component amount
2x Phusion MM 12.5 uL
MQ 9.5 uL
MBP Fw 1 uL
MBP Rev 1 uL
Colony MBP 1 uL
Touchdown MBP program Temperature Time
Denaturing 98° 2.00 min
Touchdown 10X 60->50
Denaturing 98° 30 sec
Annealing 60°->50° 30 sec
Elongation 72° 30 sec
End cycles
Start Cycles 25X
Denaturing 98° 30sec
Annealing 60° 30 sec
Elongation 72° 30 sec
End cycles
Final elongation 72° 7 min
Hold Forever
  • Overnight culture MBP

Vectors

Dry

We had a look at ways to measure things concerning gas vesicles (buoyant density, volume, etc.), buoyant density was previously measured using a specific apparatus which we're not likely to obtain. We also talked with some biologists about ways to detect protein concentrations (or rather, amounts) using antibodies, apparently this might be possible if we attach something for which there is a kind of "standard" antibody available (and the maltose binding protein we're attaching to ArsR might qualify as such).


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