Regulatory
Part:BBa_K530003:Design
Designed by: Daniel Wolozny Group: iGEM11_Johns_Hopkins (2011-08-04)
KRE9 Yeast Promoter
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 311
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Promoter was extracted from purified genomic DNA from Saccharomyces Cerevisiae. The PCR reaction performed also served to add the biobrick prefix and suffix to the promoter. PCR and tube contents This PCR was done with the use of Herculase Enzyme from Agilent.
| Reagents | Volume(uL) |
|---|---|
| Herculase 5X Buffer | 10 |
| 2.5mM dNTP Mix | 5 |
| 100-300ng DNA | X |
| 10uM Forward Primer | 1.25 |
| 10uM Reverse Primer | 1.25 |
| Herculase II Enzyme | 1 |
| Sterile Water | Fill to total |
| Total | 50 |
| Temperature (C) | Time | Cycles |
|---|---|---|
| 95 | 2 mins | 1 |
| 95 | 20 secs | 30 |
| 55 | 20 secs | 30 |
| 72 | 30 secs | 30 |
| 72 | 3 mins | 1 |
| 4 | Hold | 1 |
5ul of the sample were run on a gel with loading dye. The resulting gel confirmed the product was of the right size (541bp) to be the insert with the biobrick prefix and suffix now attached.
The insert was then PCR purified and eluted with 10mM Tris HCl at ph7.4. Following the PCR purification, the inserts were digested with EcorI and SpeI.
| Reagent | Volume(uL) |
|---|---|
| NEB Buffer 4 | 3 |
| Insert | 25 |
| EcorI-HF | 1 |
| SpeI | 1 |
| BSA | .3 |
Source
http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=S000003710