Flag Counter

Lab Notes: September

Date Notes
Sept 1PCR amplifications of lgt, DsbA-BLF1, DsbA-BLF2, addA stand, pSB1C3 linear backbone, GFP double terminator, and atrazine stand.
Sept 2Cut BBa_K411003 with E+P. Make up new competent cells with LB and glycerin. Transformation with parts in iGEM kits (BBa_K091111, BBa_K091112 in 2012 distribution, and BBa_K091112 in 2011 distribution).
Sept 3PCR amplification of theo-stand, atw-stand, DsbA-FA-lgt, DsbA-FB-lgt, and GFP with terminator.
Sept 4Find that the transformation of lacIq and placIQ1 are both successful. Receive plasmids with TrzN degradation gene from NJU.
Sept 5PCR amplification of RFP, antiholin, Trz N, holing, BLF1, and BLF2. Small-scale plasmid purification of cheZ and pSB1A3.
Sept 6Small-scale plasmid purification of FA, ribo-cheZ, and atr riboswitch. Cut PE and kanamycin with X+P.
Sept 7PCA of FB, BLF1, BLF2, and cheZ-RFP-antiholin. CPEC of DsbA-FA-lgt. Direct CPEC of DsbA-FA-lgt, DsbA-FB-lgt, DsbA-BLF1-lgt, DsbA-BLF2-lgt, and cheZ-RFP-antiholin.
Sept 8PCR amplification of lgt and DsbA-BLF1. CPEC of ptactamaze-lgt complex.
Sept 9PCR of holing, BLF2, theo, lact, and FB.
Sept 10Pick up and test four colonies of the UC Davis strain, and inoculate to LB medium. PCR amplification of BLF1-pSB1C3 and FB.
Sept 11New method: cover E. coli with calcium carbonate and calcium acid phosphate shell (safety issues). Find that E. coli in such shell can express GFP. PCR verification of anti-Dter, BLF1, BLF2.
Sept 12Redo some steps of yesterday’s PCR.
Sept 13Cut and link pluxR and cheZ; PCR clean-up. Small-scale plasmid purification of holin and K (mutant). Cut RFP with E+X, pluxR-cheZ with E+S.
Sept 14PCR amplification of BLF1, PCA-theo, and PCA-addA. Cut pSB1CB and BLF1 with E+B.
Sept 15Link pluxR-cheZ-RFP and BLH-pSB1C3. Transform addA, FA, FB, BLF1, BLF2, β-tactamase, and BLF1-1C3. Help UC Davis do transform works of three promoters in 2012 kit (BBa_J23108, BBa_J23109, and BBa_J23111).
Sept 16Pick up monoxenie of B. subtilis and inoculate.
Sept 17Find that the transformation of 2012 kit was failed. 3A assembly of luxR-cheZ, RFP, and pSB1A3.
Sept 18Cut II with N+X, III with N+P. PCR amplification of cheZ, I, lgt, and lgt-RAW.
Sept 19Mid-autumn festival. One-day holiday!
Sept 20PCR amplification of lgt-RAW, BBa_K411003, lgt-S, and shrep-back.
Sept 21Pick up five tubes of colonies and do PCR. Run gel, take photo (on computer). Transform K into BL21 (kanamycin resistance). Transform three parts again for UC Davis.
Sept 22Pick up two tubes of Theo Ribo, Atra Ribo, and A+ each and put in shaker in 37°C. PCR amplification of FA, FB, I, and lgt-RAW.
Sept 23Redo some steps of PCR yesterday.
Sept 24Find that the transformation of FB2 was failed, and FB1 successful. Cut to verify FB1 and lgB1.
Sept 25Measure the concentration curve of E. coli. Make up new Amp and Chl solution. Pick up three tubes of colonies with holin.

Next: Protocol