IGEM:Cambridge/2008/Notebook/Bacillus/2008/09/09

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(Chromosomal DNA extraction)
(Chromosomal DNA extraction)
Line 38: Line 38:
* Lane2 : AmyE Back
* Lane2 : AmyE Back
* Lane3 : AmyE Front
* Lane3 : AmyE Front
-
* Lane4 : Duncan's DNA
+
* Lane4 : Bacillus chromosomal DNA (given by Duncan to check)
* Lane5 : ECE112 (plasmid)
* Lane5 : ECE112 (plasmid)
* Lane6 : ECE153 (plasmid)
* Lane6 : ECE153 (plasmid)
Line 52: Line 52:
- New PCR with 4μL of chromosomal DNA and new PCR program
- New PCR with 4μL of chromosomal DNA and new PCR program
 +
 +
- New gel
 +
 +
* Lane1 : IA751 (chromosomal DNA)
 +
* Lane2 : ECE153 (chromosomal DNA)
 +
* Lane3 : ECE112 (chromosomal DNA)
 +
* Lane4 : ECE112 (plasmid)
 +
* Lane5 : ECE153 (plasmid)
 +
* Lane6 : Bacillus chromosomal DNA (given by Duncan)
 +
* Lane7 : HyperladderI
 +
* Lane8 : agrA
 +
* Lane9 : agrB
*Result
*Result
 +
 +
[[Image:photos09092.gif|500px|center]]
 +
 +
- No insert in IA751 transformed with ECE153 and ECE112 (same size than control!) and however the amylase test is positive for this colony....
==PCR AmyE again==
==PCR AmyE again==

Revision as of 06:43, 16 September 2008



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Chromosomal DNA extraction

- ZR soil microbe DNA kit for

  • IA751
  • IA751+ECE112
  • IA751+ECE153

- Nanodrop

product (chromosomal DNA) concentration (ng/μL)
IA751 75
IA751 + ECE112 48.6
IA751 +ECE153 74.2


- PCR with AmyE detect primers (12μL of SDW, 5μL of MM, 1+1μL of each primer, 1μL of chromosomal DNA or plasmid)

- Gel

  • Lane1 : HyperladderI
  • Lane2 : AmyE Back
  • Lane3 : AmyE Front
  • Lane4 : Bacillus chromosomal DNA (given by Duncan to check)
  • Lane5 : ECE112 (plasmid)
  • Lane6 : ECE153 (plasmid)
  • Lane7 : chromosomal DNA IA751 + ECE112
  • Lane8 : chromosomal DNA IA751 + ECE153
  • Lane9 : chromosomal DNA IA751
  • Results

- Only something for IA751, and ECE153 (pure plasmid)???

- New PCR with 4μL of chromosomal DNA and new PCR program

- New gel

  • Lane1 : IA751 (chromosomal DNA)
  • Lane2 : ECE153 (chromosomal DNA)
  • Lane3 : ECE112 (chromosomal DNA)
  • Lane4 : ECE112 (plasmid)
  • Lane5 : ECE153 (plasmid)
  • Lane6 : Bacillus chromosomal DNA (given by Duncan)
  • Lane7 : HyperladderI
  • Lane8 : agrA
  • Lane9 : agrB
  • Result

- No insert in IA751 transformed with ECE153 and ECE112 (same size than control!) and however the amylase test is positive for this colony....

PCR AmyE again

- 24μL of SDW, 10μL of MM, 2μL of each primers, 2μL of ECE112

  • Results (cf gel, lane 2 and 3)

- Very good result for amyE front, nothing for AmyE back


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