Team:Warsaw/Calendar-Main/27 August 2009

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Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil


Tasks:

  • Amplification of the mgtc promoter using new primers
  • Isolation of the pSB1A3 plasmid

Methods:

  • The PCR mix was prepared as follows:

    5μl buffer B (EURx)
    5μl 10mM dNTPs
    5μl forward starter
    5μl reverse starter
    2μl OptiTaq polymerase (EURx)
    2μl Salmonella matrix
    26 μl H2O
  • PCR programme:

     4min 95°C 
    (30s 95°C, 35s 56°C, 40s 72°C)x28
    10min 72°C
    ~ 4°C
  • The PCR results were visualised with gel electrophoresis on 1% agarose gel.
  • The plasmid isolation was carried out from 3μl of culture cultivated for 6h in 37°C. The Plasmid Mini kit (A&A Biotechnology) was used.

Results:

  • Nothing

Conclusions:

  • The temperature was chosen... poorly.

Cloning of the cro-box into the pSB1A3 plasmid

Kamil


Tasks:

  • Creation of the cro-box double stranded DNA

Methods:

  • The mixture of equal volumes (10μl) of both ssDNA was heated to 94°C for 15min. and left to cool.

Assembly of endosomal detection operon

Marcin


Task 1:

Methods:

  • Reaction mixture composition:
  •  
    20 μl purified plasmid DNA product
    1 μl XbaI (Fermentas)
    1 μl PstI (Fermentas) in the case of C0051
    0.7 μl SpeI (Fermentas) in the case of E0022
    2 μl Buffer Tango (Fermentas)
    15 μl MQ water
  • Digest was performed about seven hours
  • After digestion samples were frozen

Making of the plac-RBS-llo-intA part

Jarek


Tasks:

  • Separation of digested DNA fragments in 0,8% agarose.

Results:

  • The intA gene that was used might not be the intA gene after all.

Cloning switch 1 regulatory parts [ K177012-PcI.RBS.LacI, K177033-PcI.RBS.LacI.PcI.RBS.RFP.terminator, K177011-PLacI.RBS.cI.terminator, K177038-PLacI.RBS.cI.terminator.PLacI.RBS.GFP.terminator ] into two compatible low copy number plasmids of different antibiotic resistance

Ania


Tasks:



April
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May
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June
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8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
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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
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10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
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7 8 9 10 11 12 13
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21 22 23 24 25 26 27
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October
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<center>

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
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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