Koeris/Notebook/2007-1-15: Difference between revisions
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==Preparation== | ==Preparation== | ||
What can I do in order to make sure the cloning works this time? | What can I do in order to make sure the cloning works this time? | ||
*Check concentrations of the chromatin DNA | *Check concentrations of the chromatin DNA: '''351 ng/ul''' | ||
*Do a dilution series over | *Do a dilution series over two orders of magnitude of the template DNA: 3ng/ul - 300ng/ul | ||
**All dilutions are to be done in at '''LEAST 100ul volume''' ddH<sub>2</sub>O | |||
**Dilute stock 1:1.2: yields 300ng/ul | |||
**Dilute twice 1:10: yields 30ng/ul and 3ng/ul | |||
==PCR set-up of the operon== | ==PCR set-up of the operon== | ||
#Master mix | #Master mix |
Revision as of 09:12, 15 January 2007
Pseudomonas aeruginosa mer operon cloning 3
Preparation
What can I do in order to make sure the cloning works this time?
- Check concentrations of the chromatin DNA: 351 ng/ul
- Do a dilution series over two orders of magnitude of the template DNA: 3ng/ul - 300ng/ul
- All dilutions are to be done in at LEAST 100ul volume ddH2O
- Dilute stock 1:1.2: yields 300ng/ul
- Dilute twice 1:10: yields 30ng/ul and 3ng/ul
PCR set-up of the operon
- Master mix
- Template DNA equiv.
- ddH2O to 50uL
- Thermal profile
- 92 deg C - 5min
- 92 deg C - 30s
- 50 deg C - 45s
- 72 deg C - 120s
- 4 deg C - indefinite
Pipetting scheme
Column heading 1 | Column heading 2 | Column heading 3 |
---|---|---|
Row heading 1 | Cell 2 | Cell 3 |
Row heading A | Cell B | Cell C |
Gel image
1% agarose TAE gel, ran @ 100V for 60min