IGEM:University of Groningen/2011/Notebook/Count coli/2011/08/18

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Count coli Main project description
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Thu 18 Aug 2011

To Do List

1. Ligate pSB1K3+revDT+P(LasR)+LasR and revP(BAD)-DT

  • Digest vector with SP, insert with XP--incubate 1 hr 37 degree
  • Purify and elute in 50 μl
  • Ligate ON

2. Ligate pSB1A3+RBS-RFP-DT and digested Pc

  • Digest vector with EX, insert with ES--incubate 1 hr 37 degree
  • Run digested insert on gel then extract it from 3% agarose gel
  • Purify and elute in 50 ul for vector and 20 μl for insert
  • Ligate ON

Implementation

1. Ligate 12 variants of pSB1K3+revDT+P(LasR)+LasR and revP(BAD)-DT
Double digestion
Vector --> SP
18 μl MQ
3 μl 10x buffer
1 μl RE1
1 μl RE2
1 μl FastAP
6 μl vector


insert --> XP
14 μl MQ
2 μl 10x buffer
1 μl RE1
1 μl RE2
1 μl FastAP
2 μl vector

Ligation mix [insert] = 11 ng/nl
For 1:9 vector to insert ratio, insert mass = 100 ng ~ 10 μl
2 μl 10x buffer
1 μl T4 DNA ligase
7 μl vector
10 μl insert

2. Ligate pSB1A3+RBS-RFP-DT and digested Pc
Double digestion
Vector --> EX [vector] = 43 ng/ul
20 μl MQ
3 μl 10x buffer
1 μl RE1
1 μl RE2
1 μl FastAP
4 μl vector


insert --> ES [insert] = 95 ng/ul
11 μl MQ
2 μl 10x buffer
1 μl RE1
1 μl RE2
5 μl vector

Ligation mix
2 μl 10x buffer
1 μl T4 DNA ligase
7 μl vector
10 μl insert

Highlight

  • Weekly team meeting at 2 pm
  • Update meeting with supervisor and advisor at 4 pm


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