IGEM:Cambridge/2008/Notebook/Bacillus/2008/09/09: Difference between revisions
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==Chromosomal DNA extraction== | ==Chromosomal DNA extraction== | ||
- ZR soil microbe DNA kit | - ZR soil microbe DNA kit for | ||
*IA751 | |||
*IA751+ECE112 | |||
*IA751+ECE153 | |||
- Nanodrop | |||
{|class="wikitable" style="text-align:center" border="1" | |||
|- | |||
! 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 | |||
[[Image:photosdfscontrols.gif|300px|center]] | [[Image:photosdfscontrols.gif|300px|center]] | ||
* 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 | |||
[[Image:photos09092.gif|300px|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== | |||
- 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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Revision as of 03:43, 16 September 2008
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Chromosomal DNA extraction- ZR soil microbe DNA kit for
- Nanodrop
- Gel
- Only something for IA751, and ECE153 (pure plasmid)??? - New PCR with 4μL of chromosomal DNA and new PCR program - New gel
 - 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
- Very good result for amyE front, nothing for AmyE back | |||||||||
