IGEM:MIT/2006/Notebook/2006-8-4

to do

1. make glycerols of R0040-E0840 -done
2. LC multiple transformants -done
3. colony PCR multiple transformants and run on gel-done
4. PCR BAT2 and THI3, cleanup, run on gel -done
   • note: got much higher dna concentrations this time post pcr cleanup using lab kit
5. sequence promising colony PCRs and throw out bad LCs
6. repeat 3-part assembly plan -use our own cut [A/T: EP] backbone instead of Meagan's -done
   • digest
   • ligate
   • transform
7. discuss big picture rolls/project PLAN and peoples' upcoming schedules

Transformants Found

B0030-ATF1-B0015 2 (Gel Extraction), B0030-ATF1-B0015 6 (Gel Extraction), and B0030-BSMT-B0015 (Gel Extraction)

Conclusions

1. pUC19 again did not work. We need a new control plasmid.
2. Since Meagan said that the AC and AT backbones did not work in her assembly and they did not work in our assembly, we should try to get new backbones for the antibiotic construction method. Until then, we should do assemblies using just the gel extraction method.
3. We need to check to see if the three plates contain the correct colonies. We will colony PCR today, and if we see the correct bands, we should LC, miniprep, and sequence those colonies while moving ahead with attaching R0040 to the two structures.

Colony PCR

1. B0030-BSMT-B0015
2. B0030-ATF1mut-B0015(2)
3. B0030-ATF1mut-B0015(6)

Sequencing

1. Only sequence promising colony PCR results
   • weed out BOO30 in backbone

digests (11:45)

1. pSB1AT3: EP x2
2. THI3: SX