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Team:UPO-Sevilla/Notebook/08 10
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<h2>Assembly Team</h2> | <h2>Assembly Team</h2> | ||
| - | <p | + | <p>We repeated SDM reactions for <i>fecA*</i> (UPO28) and <i>gltD**</i> (UPO17) from the beginning. This time we used <i>Taq expand enzyme</i> instead of <i>Pfu</i>. In the results, we did not obtain any product of amplification in the first SDM’s PCR reaction. Next we made the second SDM’s PCR reaction using first former SDM’s PCR reaction products. Again the results were not satisfactory.</p> |
<p>Transformation of <i>E. coli</i> DH5α with ligations made the day before.</p> | <p>Transformation of <i>E. coli</i> DH5α with ligations made the day before.</p> | ||
Revision as of 20:01, 23 October 2010
August, 10th
Assembly Team
We repeated SDM reactions for fecA* (UPO28) and gltD** (UPO17) from the beginning. This time we used Taq expand enzyme instead of Pfu. In the results, we did not obtain any product of amplification in the first SDM’s PCR reaction. Next we made the second SDM’s PCR reaction using first former SDM’s PCR reaction products. Again the results were not satisfactory.
Transformation of E. coli DH5α with ligations made the day before.
We analyzed BamI-PstI digestions in 1% agarose gel: colony 4 and 5 are positive.
The 2+28 and 28+3 day before transformation plates have not white colonies. 7+2+28 colonies are strange. We will repeat these devices using low copy vectors. We set up inocula of positive candidate colonies.
Preparative digestion of UPO 4, 5 and 11 (in Mr. Gene vector) and 1% agarose gel. GFX purification. Ligation with pSB1C3 and transformation.
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