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User:University of Washington/30 July 2008
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| + | == Lambda Red Recombineering of RP4 (Bryan) == | ||
| + | Co-transformed DY331 with pUC307 (RP1) and recombinant vector containing Cm resistance cassette. Time constants 1.0 and 1.2 for two trials. Suspect glycerol stocks may contribute to poor time constant. Plated on selective media. | ||
| + | Since I expect poor transformation, innoculated ON culture of DY331 for next transformation experiment. DY331 will be rendered electrocompetent and immediately transformed to eliminate any problems that may be due to using a glycerol stock. | ||
| + | |||
| + | ==AHL Expression in Yeast (Bryan)== | ||
| + | |||
| + | X & S double restriction digest on yeast expression plasmids and C0161. Ran on gel. Analysis pending. | ||
| + | |||
| + | ==LuxR from AraC and TetR== | ||
| + | |||
| + | - Did restriction digestion on Elowitz's plasmid with HindIII. (30 ul of DNA for digestion reaction, 15 ul for control with no enzyme). Incubated the reaction for about 4.5 hours, then ran the gel. Saw bands: 4-5 kb, 2 kb, 0.5-1 kb as expected. However, the 4-5 kb fraction wasn't clear enough to tell whether it was only 4.7kb of pCD26 or 4.2kb of pACYC184. Will have to select another enzyme to confirm the nonexistence of pACYC184(this produces LuxR which we don't want). | ||
| + | |||
| + | - Overnighted Elowitz's plasmid in Tsy+Kan, E0240 in Tsy+Amp. | ||
| + | |||
| + | - Streaked out Elowitz's E.coli in Tsy plate. | ||
| + | |||
| + | |||
| + | ==LuxR from pLac== | ||
| + | |||
| + | -Restriction digests of parts E0240 and R0010 were ran on gel for isolation. However, no DNA lines were seen in the rescrition lanes of the gel. A possible explaination is that the enzymes were left to incubate with the DNA too long (overnight), and chewed up all the DNA. | ||
| + | |||
| + | -Another restriction digest of ROO10 and E0240 was started for run overnight. | ||
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Latest revision as of 19:13, 5 August 2008
Contents |
Lambda Red Recombineering of RP4 (Bryan)
Co-transformed DY331 with pUC307 (RP1) and recombinant vector containing Cm resistance cassette. Time constants 1.0 and 1.2 for two trials. Suspect glycerol stocks may contribute to poor time constant. Plated on selective media.
Since I expect poor transformation, innoculated ON culture of DY331 for next transformation experiment. DY331 will be rendered electrocompetent and immediately transformed to eliminate any problems that may be due to using a glycerol stock.
AHL Expression in Yeast (Bryan)
X & S double restriction digest on yeast expression plasmids and C0161. Ran on gel. Analysis pending.
LuxR from AraC and TetR
- Did restriction digestion on Elowitz's plasmid with HindIII. (30 ul of DNA for digestion reaction, 15 ul for control with no enzyme). Incubated the reaction for about 4.5 hours, then ran the gel. Saw bands: 4-5 kb, 2 kb, 0.5-1 kb as expected. However, the 4-5 kb fraction wasn't clear enough to tell whether it was only 4.7kb of pCD26 or 4.2kb of pACYC184. Will have to select another enzyme to confirm the nonexistence of pACYC184(this produces LuxR which we don't want).
- Overnighted Elowitz's plasmid in Tsy+Kan, E0240 in Tsy+Amp.
- Streaked out Elowitz's E.coli in Tsy plate.
LuxR from pLac
-Restriction digests of parts E0240 and R0010 were ran on gel for isolation. However, no DNA lines were seen in the rescrition lanes of the gel. A possible explaination is that the enzymes were left to incubate with the DNA too long (overnight), and chewed up all the DNA.
-Another restriction digest of ROO10 and E0240 was started for run overnight.
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