IGEM:MIT/2006/Notebook/2007-1-11

Plating Results from Yesterday

1. Jason's parts finally grew up. The control, B0015, worked only on the Kan plate as expected (it is in the 1AK3 plasmid). Mach I interestingly grew up on Kan and Tet but not Chl. pBANANA #10 and #12 both grew up on Kan and Chl but not on Tet.

2. J45180 only grew up on AMP/KAN as expected, confirming that the cell is unlikely to have plasmids of different resistances in it.

3. Interestingly, J45400, which was streaked out by Meagan on an AMP/KAN plate did not grow on AMP/KAN, AMP/TET, or AMP/CHL. I will repeat this experiment today.

4. Many colonies were seen from the transformations of J45120, the two J45800 hopefuls, and pUC18.

Plan Today

1. Miniprep, glycerol, and sequence J45400- DONE

2. Repeat streaking of J45400 onto AMP/KAN, AMP/TET, and AMP/CHL- DONE

3. Write UROP Proposal- DONE

4. Perform colony PCR on many colonies from the J45120 and J45800 transformations- DONE and make LCs- DONE

5. Track down Indole-Knockout cells- STILL LOOKING

6. Check for sequencing results- DONE

7. Make a checklist for each part- DONE

8. Run gels from the colony PCR of J45120 and J45800 transformants - J45120: most colonies were approx 1600 kB on the gel, which is correct. will read the gel for J45800 tomorrow

SEQUENCING RESULTS

• J45700 only had J45320 in it, but no J45120
• J45320 looked correct
• J45180 was just the osmY + inverter, except no R0040 + WGD
• J45200 looked correct, but the internal forward primer didn't work so we didn't get sequencing data for approx 150 base pairs in the middle. we'll do a smell test to make sure it's correct
• J45120 had an AAT insertion between 1197 and 1198, which should be ok. we'll do a smell test for that too.