Team:Warsaw/Calendar-Main/8 September 2009

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* Seting ligation of internaline A with pUC18.
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<h3><div style="text-align: center;">Inverting PLacI regulated parts of the switch and assembling the switch on a single plasmid<h3>
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<h4>Ania</h4>
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Revision as of 01:20, 10 September 2009


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Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil


Tasks:

  • Colonies transfer
  • Liquid cultures establishment

Methods:

  • Appropriate colonies (all 5 of them) were transferred to a new plate containing ampicilin.
  • Liquid cultures were established in 4 ml LB medium supplemented with ampicilin and incubated overnight with areation.

Cloning of the cro-box into the pSB1A3 plasmid

Kamil


Tasks:

  • Colonies transfer
  • Liquid cultures establishment

Methods:

  • Appropriate colonies (all 7 of them) were transferred to a new plate containing ampicilin.
  • Liquid cultures were established in 4 ml LB medium supplemented with ampicilin and incubated overnight with areation.


Contents

Assembly of endosomal detection operon

Marcin

Task 1: Isolation of following construct from liquid bacterial culture:

Methods:

  • Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here

Task 2:

  • Digest previously isolated plasmid to verify the correctness of the ligation:

Methods:

  • Reaction mixture composition:
2 μl purified plasmid DNA product
1 μl PstI (Fermentas)
1 μl XbaI (Fermentas)
2 μl Buffer Tango (Fermentas)
15 μl MQ water
  • Digest was performed 3 hours

Results:

Task 3:

Methods:

  • Reaction mixture composition:
1. μl purified plasmid DNA product
1 μl XbaI (Fermentas)
1 μl PstI (Fermentas) 
2 μl Buffer Tango (Fermentas)
15 μl MQ water
  • Digestions were carry out 5 hours and subsequently were inactivated via heating for 20 minutes

Comment: The length of digested sequence is unexpected. The correct sequence should have had 1500 bp. Althought in each case the length is approximately 950 bp. It is obligated to carry out sequencing reaction to reveal the identity of this DNA.


Assembly of fusion proteins

Marcin

Task:1

  • Transformation of chemocompetent E. coli TOP10 strain

Construct to transform:

  • signal peptid from Cox1 CDS on pKSII vector

Methods:

  • Ligation mixture was thermally inactivated in 65 °C
  • Detailed protocol of transformation is described here

Acquiring the internaline A gene from Listeria monocytogenes st. EDG-e genome

Jarek

Tasks:

  • Phosphorylation of PCR product with T4 phage kinase.
  • Digestion of pUC18 with HincII endonuclease and dephosphorylation of it's ends with CIAP phosphatase.
  • Seting ligation of internaline A with pUC18.


Inverting PLacI regulated parts of the switch and assembling the switch on a single plasmid

Ania


Tasks:


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