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

{| width="800"
 * style="background-color: #444444"|[[Image:Bacillus_button.gif|200px|center]]
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Chromosomal DNA extraction
- ZR soil microbe DNA kit for
 * IA751
 * IA751+ECE112
 * IA751+ECE153

- Nanodrop

- 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


 * }