Team:Groningen/Notebook/15 July 2009

From 2009.igem.org

(Difference between revisions)
(Transporters)
(Vectors)
 
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* Cells added to 3 ml Ty in a reaction tube
* Cells added to 3 ml Ty in a reaction tube
* Incubation 1 h in waterbath shaker 37°C
* Incubation 1 h in waterbath shaker 37°C
 +
 +
* The cells are plated on Ty agar (100 μg/ml)
 +
* On one plate 200 μL is streaked out. The rest is concentrated (centrifugation: 1 min, 14600rpm) in 200 μL and also plated.
 +
* Incubation o/n 37°C
===Transporters===
===Transporters===
-
We decided to make MasterMixes of our own. All but template, primers and taq.
+
We decided to make MasterMixes of our own. All but 1uL template, 2uL primers and 1uL taq for 25uL reactions.
{|
{|
|
|
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{| border="1"
{| border="1"
|+ '''1X MasterMix Buffer NH SO'''
|+ '''1X MasterMix Buffer NH SO'''
-
! Component  
+
! Component
 +
! concentrations
 +
! volumes
|-
|-
! dNTP  
! dNTP  
-
| 2.0 mM   
+
| 0.2 mM   
|8 uL
|8 uL
|-
|-
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|80 uL
|80 uL
|-
|-
-
!taq buffer (NH)SO
+
!taq buffer KCl
|1X
|1X
|100 uL
|100 uL
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|652 uL
|652 uL
|}
|}
-
 
-
 
|width="10"|
|width="10"|
|
|
<!--Tabel 2 hier-->
<!--Tabel 2 hier-->
 +
{| border="1"
{| border="1"
|+ '''1X MasterMix Buffer KCl'''
|+ '''1X MasterMix Buffer KCl'''
-
! Component  
+
! Component
 +
! concentrations
 +
! volumes
|-
|-
! dNTP  
! dNTP  
-
| 2.0 mM   
+
| 0.2 mM   
|8 uL
|8 uL
|-
|-
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|672 uL
|672 uL
|}
|}
 +
 +
|width="10"|
 +
|
 +
<!--Tabel 3 hier-->
 +
|}
 +
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|
|
<!--Tabel 3 hier-->
<!--Tabel 3 hier-->
 +
|}
 +
 +
==Vectors==
 +
* overnight cultures were diluted 100, 200 or 400 x
 +
* 200 ul of culture per well
 +
* Measured for 16 h
 +
:: - every 15 min
 +
:: - @ 37&deg;  with shaking 6 mm
 +
:: - for mRFP1  excitation =590 nm
 +
::              excitation =610 nm
 +
 +
 +
 +
{| border="1"
 +
|+ '''96-wells Microtiter plate'''
 +
!
 +
! 1 LB
 +
! 2 LB
 +
! 3 LB
 +
! 4 LB
 +
! 5 LB
 +
! 6
 +
! 7 TY
 +
! 8 TY
 +
! 9 TY
 +
! 10 TY
 +
! 11 TY
 +
! 12
 +
|-
 +
! A 100x
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
|-
 +
! B 100x
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
|-
 +
! C 200x
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
|-
 +
! D 200x
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
|-
 +
! E 400x
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
|-
 +
! F 400x
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
|-
 +
! G
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|
 +
 +
|-
 +
! H
 +
|
 +
|
 +
|
 +
|
 +
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 +
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|}

Latest revision as of 12:45, 17 August 2009

Igemhomelogo.png

Wet

GVP Cluster

Restriction for Assembly

The vector (BBa_J61035) containing GVP cluster was cut with PstI and XbaI to create correct ends for insert in promotor vector (BBa_J61002).

  • 10μL MQ
  • 6μL plasmid in MQ (2μg) (329.7 ng/μL)
  • 2μL Fast digest buffer
  • 1μL PstI fast digest enzyme
  • 1μL XbaI fast digest enzyme

The vector (BBa_J61002) containing promotor BBa_J23100 was cut with SpeI and PstI.

  • 4μL MQ
  • 12μL plasmid in MQ (155.4 ng/μL)
  • 2μL Fast digest buffer
  • 1μL SpeI fast digest enzyme
  • 1μL PstI fast digest enzyme

The cups were incubated in a heat block at 37°C for 30 min. followed by adding 4μL 6x loading buffer for gel electroforesis.


Gel electroforesis

24μL of each sample was loaded on a 1% agarose gel (devided over two slots) with EtBr and a 1kb ladder was used (see picture).

Gel 2009-07-15.jpg Generulers 1kb marker Fermentas.jpg

→ From left to right: Empty slot, 1kb Marker, Empty slot, HmtA PCR reaction, Empty slot, (2x) GVP, Empty slot, (2x) BBa_J23100, and Empty slot


Gel Purification of GVP

"High Pure PCR Product Purification Kit" from Roche was used for gel purification.

  • ~200mg of gel containing the desired fragment was dissolved in 500μL binding buffer by heating to 60°C for 10 min. and carefully vortexing.
  • 250μL isopropanol was added to the tube and vortexed
  • dissolved gel was transfered to the column and centrifuged for 1 min.
  • column was washed with 500μL Wash Solution, centrifuged for 1 min., 200μL Wash solution was added and centrifuged 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-cluster eluted in MQ

  • 33.5 ng/μL
  • 1.65 (260/280)
  • 0.77 (260/230)

BBa_J23100 + vector eluted in MQ

  • 14.2 ng/μL
  • 1.78 (260/280)
  • 1.01 (260/230)
→ Concentrations are just high enough for a ligation reaction to be performed, some additional tuning of the procedure is required!!


Ligation of GVP into BBa_J23100 vector

Mix:

  • 1 μL T4 Ligase buffer
  • 1 μL Vector (J23100 restriction fragment, 14.2 ng/μL)
  • 7.6 μL Insert (GVP gene cluster, 33.5 ng/μL)
  • 0.5 μL T4 Ligase

Method:

  • The mixture is incubated 30 min, 26°C
  • To inactivate Ligase 10 min 70°C


Transformation of E.coli TOP10 cells with GVP + J23100

  • 2 μL Ligation mix added to cells from -80°C glycerol stock
  • 30 min incubation on ice
  • 5 min heat shock 37°C
  • 5 min on ice
  • Cells added to 3 ml Ty in a reaction tube
  • Incubation 1 h in waterbath shaker 37°C
  • The cells are plated on Ty agar (100 μg/ml)
  • On one plate 200 μL is streaked out. The rest is concentrated (centrifugation: 1 min, 14600rpm) in 200 μL and also plated.
  • Incubation o/n 37°C


Transporters

We decided to make MasterMixes of our own. All but 1uL template, 2uL primers and 1uL taq for 25uL reactions.

1X MasterMix Buffer NH SO
Component concentrations volumes
dNTP 0.2 mM 8 uL
MgCl 2.0 mM 80 uL
taq buffer KCl 1X 100 uL
MQ 652 uL
1X MasterMix Buffer KCl
Component concentrations volumes
dNTP 0.2 mM 8 uL
MgCl 1.5 mM 60 uL
taq buffer (NH)SO 1X 100 uL
MQ 672 uL


MasterMix3
Component Mg/K
MQ 28.5 uL
F mut1 2 uL
R mut2 2 uL
dNTP 2 uL
MgCl 3 uL
taq buffer KCl 5 uL
taq buffer (NH)SO 5 uL
taq polymerase 2.5 uL
PCR 3 program Temperature Time
Denaturing 95° 2.00 min
Start Cycles 25X
Denaturing 95° 30 sec
Annealing 55° 20 sec
Elongation 72° 2.10 min
End cycles
Final elongation 72° 10 min
Hold Forever

Vectors

  • overnight cultures were diluted 100, 200 or 400 x
  • 200 ul of culture per well
  • Measured for 16 h
- every 15 min
- @ 37° with shaking 6 mm
- for mRFP1 excitation =590 nm
excitation =610 nm


96-wells Microtiter plate
1 LB 2 LB 3 LB 4 LB 5 LB 6 7 TY 8 TY 9 TY 10 TY 11 TY 12
A 100x pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100 pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100
B 100x pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100 pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100
C 200x pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100 pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100
D 200x pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100 pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100
E 400x pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100 pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100
F 400x pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100 pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100
G
H

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