Team:Groningen/Notebook/17 July 2009

From 2009.igem.org

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{|
{|
! PCR1 program  
! PCR1 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
 +
|4°
 +
|Forever
 +
|}
 +
|width="10"|
 +
|
 +
<!--Tabel 3 hier-->
 +
|}
 +
 +
 +
{|
 +
|
 +
<!--Tabel 1 hier-->
 +
 +
{| border="1"
 +
|+ '''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
 +
|-
 +
|}
 +
 +
|width="10"|
 +
|
 +
<!--Tabel 2 hier-->
 +
{|
 +
! 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
 +
|4°
 +
|Forever
 +
|}
 +
|width="10"|
 +
|
 +
<!--Tabel 3 hier-->
 +
|}
 +
 +
{|
 +
|
 +
<!--Tabel 1 hier-->
 +
 +
{| border="1"
 +
|+ '''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
 +
|-
 +
|}
 +
 +
|width="10"|
 +
|
 +
<!--Tabel 2 hier-->
 +
{|
 +
! PCR 3 program
! Temperature
! Temperature
! Time
! Time

Revision as of 14:17, 17 July 2009

Igemhomelogo.png

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


Wet

GVP Cluster

Discussion:

All colonies (transformants vector + promoter ligated with gvp) and o/n culture became red, so probably RFP is still in there... What might be the problem? The vector with promoter self-ligated due to uncomplete digestion (done 15 July 2009) leading to the following fragments:

  • vector+RFP linear
  • vector
  • RFP

When the upper fragment (~2kb) is contaminated with linear vector this would lead high efficiency self-ligation instead of ligating gvp with the emtpy vector.

→TODO Therefore the restriction/purification/ligation will be redone and selection of non-red colonies will be made!
→DONE Also plasmid from the o/n cultures will be purified and analysed by restriction analysis.
  • For plasmid isolation the Sigma Plasmid isolation kit was used (eluted in 50ul MQ).
  • DNA concentrations:
Sample ng/ul 260/280 260/230
gpv-promoter test (colony 9) 82.5 2.02 2.21
gpv-promoter test (colony 10) 71.9 1.96 2.21
  • Restriction analysis with SpeI and PstI to find insert length.
    • Expected length with RFP insert ~1kb
    • Expected length with gvp insert ~7kb

Restriction mixture for J23100+gvp (fragment sizes: 2096 (promoter + plasmid backbone), 887)

  • 10 μL promoter plasmid (61.4 ng/μL, 96.1 ng/μL)
  • 6μL MQ
  • 2μL Fast digest buffer
  • 1μL PstI fast digest enzyme
  • 1μL SpeI fast digest enzyme

Transporters

PCR15-7-19.jpg

Because of the negative results we will try to get positive results with an other polymerase enzyme; Phusion. And try the cloning again as planned. PCR1 Fw,mut1RC should give product of 1153bp and PCR2 Rev,mut2RC a 261bp size product.

MasterMix1
Component amount
2x Phusion MM 12.5 uL
MQ 9.5 uL
F mut1 1 uL
R mut2 1 uL
DNA 1 uL
PCR1 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 4° Forever


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 4° Forever
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 4° Forever

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
Retrieved from "http://2009.igem.org/Team:Groningen/Notebook/17_July_2009"