Team:Groningen/Notebook/17 July 2009

From 2009.igem.org

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|+ '''MasterMix3'''
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| align="center" style="background:#f0f0f0;"|'''PCR 16-07'''
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! Component !! Mg/K
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| align="center" style="background:#f0f0f0;"|'''Sample names'''
 +
| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|''''''
|-
|-
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! MQ
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| Component||B1||B2||C1||C2||A1||A2||E1||E2||F1||F2
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| 28.5 uL
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|-
|-
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! F mut1
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| MM||21||22||21||22||21||22||21||22||21||22
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| 2 uL
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|-
|-
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! R mut2
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| F 1uL||Fw||Fw||Fw||Fw||mut1||mut1||Rev||Rev||Fw||Fw
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| 2 uL
+
|-
|-
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! dNTP
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| R 1uL||Rev||Rev||mut2||mut2||PCR2||PCR2||mut1||mut1||mut1RC||mut1RC
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| 2 uL
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|-
|-
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! MgCl
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| DNA 1uL||1||0||1||0||1||0||1||0||1||0
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| 3 uL
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|-
|-
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! taq buffer KCl
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| taq 1uL||1||1||1||1||1||1||1||1||1||1
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| 5 uL
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|-
+
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! taq buffer (NH)SO
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| 5 uL 
+
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|-
+
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! taq polymerase
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| 2.5 uL
+
|-
|-
 +
|
|}
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|width="10"|
 
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|
 
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<!--Tabel 2 hier-->
 
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{|
 
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! PCR 3 program
 
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! Temperature
 
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! Time
 
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|-
 
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|Denaturing
 
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|95°
 
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|2.00 min
 
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|-
 
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|
 
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|Start Cycles 25X
 
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|-
 
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|Denaturing
 
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|95°
 
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|30 sec
 
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|-
 
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|Annealing
 
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|55°
 
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|20 sec
 
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|-
 
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|Elongation
 
-
|72°
 
-
|2.10 min
 
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|-
 
-
|
 
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|End cycles
 
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|-
 
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|Final elongation
 
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|72°
 
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|10 min
 
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|-
 
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|Hold
 
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|4°
 
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|Forever
 
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|}
 
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|width="10"|
 
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|
 
<!--Tabel 3 hier-->
<!--Tabel 3 hier-->
{|
{|

Revision as of 10:36, 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.
  • Used Sigma Plasmid isolation kit.
  • DNA concentrations:

Transporters

PCR 16-07 Sample names ' ' ' ' ' ' ' ' '
ComponentB1B2C1C2A1A2E1E2F1F2
MM21222122212221222122
F 1uLFwFwFwFwmut1mut1RevRevFwFw
R 1uLRevRevmut2mut2PCR2PCR2mut1mut1mut1RCmut1RC
DNA 1uL1010101010
taq 1uL1111111111
PCR15-7-19.jpg

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"