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User:University of Washington/25 July 2008
From 2008.igem.org
LuxR from AraC and TetR
-Received primers to biobrick Elowitz's plasmid.
-Did PCR amplification on Elowitz's plasmid (from 4 minipreps).
- Reaction Set-Up
| reagents | amount per reaction(ul) |
|---|---|
| sterile dH2O | 34.5 |
| buffer for Taq (-MgCl2) | 5 |
| 10 mM dNTPs | 1 |
| MgCl2 | 2 |
| F primer(#436) | 2.5 |
| R primer(#436) | 2.5 |
| Taq | 0.5 |
| DNA template | 2 |
- Thermocycling
| Segment | Cycles | Temperature | Time |
|---|---|---|---|
| 1 | 1 | 95°C | 1 minute |
| 2 | 30 | 95°C | 30 seconds |
| 50°C | 1 min | ||
| 72°C | 1 min | ||
| 3 | 1 | 72°C | 7 minutes |
| 4 | - | 4°C | infinite |
- Good news. Ran gel on PCR product. Expected 172 bp. Got fragments shorter than 500 kb. =]
- Bad news. Sequencing result for second trial Quikchange came. The sequences did not show any mutation. =[
LuxR from pLac
-R0010+E0420 transformed cells' DNA was sent in for sequencing.
-Glycerol stock of DH5a+lacq strain made.
-One aliquot of electrocompetent DH5a+lacq cells were made.
-Sequence of part I0462 from the 2007 plates was received back and found to be the incorrect sequence. Sent an email to igem hq requesting bacterial stab of part I0462.
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