Team:Lethbridge/Notebook/Lab Work/September

From 2010.igem.org

(Difference between revisions)
(→September 23, 2010)
Line 198: Line 198:
<BLOCKQUOTE>
<BLOCKQUOTE>
 +
 +
==<font color="white">September 21, 2010==
 +
(ADS)<br>
 +
<b>Objective:</b> Insert xylE (with N and C terminal fusion standards, obtained by PCR of K118021 by KG) into pSB1C3 plasmid for submission to registry.<br>
 +
 +
<b>Method:</b>
 +
*Restriction of xylE PCR product and pSB1C3 (containing J04450 biobrick) via BioBrick Method using EcoRI-HF and PstI (Enzymes from NEB)
 +
*Ligation of xylE PCR product and pSB1C3 via BioBrick Method using T4 DNA ligase (Enzyme from NEB)
 +
*Transform into Library Efficiency Compentent DH5&alpha; Cells (Invitrogen)
 +
 +
<b>Results:</b>
 +
*Obtained too numerous to count (TNTC) colonies.
 +
**Obtained ~100 white colonies (indicating removal of RFP and insertion of new BioBrick)
 +
==<font color="white">September 23, 2010==
==<font color="white">September 23, 2010==

Revision as of 01:51, 25 September 2010



Feel free to look around our notebook!

Here you can check out the work we have done in the lab, click on a month to take a look!

September 21, 2010

(ADS)
Objective: Insert xylE (with N and C terminal fusion standards, obtained by PCR of K118021 by KG) into pSB1C3 plasmid for submission to registry.

Method:

  • Restriction of xylE PCR product and pSB1C3 (containing J04450 biobrick) via BioBrick Method using EcoRI-HF and PstI (Enzymes from NEB)
  • Ligation of xylE PCR product and pSB1C3 via BioBrick Method using T4 DNA ligase (Enzyme from NEB)
  • Transform into Library Efficiency Compentent DH5α Cells (Invitrogen)

Results:

  • Obtained too numerous to count (TNTC) colonies.
    • Obtained ~100 white colonies (indicating removal of RFP and insertion of new BioBrick)


September 23, 2010

( JV)

Objective: Characterized catechol degradation by xylE enzyme

Method: Measured absorbance of catechol (275nm) and 2-hydroxymuconate semialdehyde (380nm).

  • Protocol:
  • 1) Grow cells in M9 minimal medium
  • 2) Take 1/10 dilution of cells
  • 3) Introduce 1µL of 0.05M catechol solution into the cell dilution. (Final concentration of 50&microM;).
  • 4) Quench the reaction with 5A% w/v trichloroacetate at certain time points. (0,15sec, 30sec, 45sec, 60sec, 2min, 3min, 4min, 5min, 10min).
  • 5) Spin down cells.
  • 6) Measure absorbance of supernatant.

Results:

Retrieved from "http://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/September"