Team:UNIPV-Pavia/Notebook/Week1

Week 1: 05/19/08 - 05/23/08
05/19/08
 * Let’s start our IGEM 2008 experience! At first, we broke the punch tool…:)


 * We used a scalpel to cut and resuspend the following 22 paper spots:


 * We used tweezers to put the cut paper into tubes containing 10 μl of warmed TE buffer.


 * We transformed 60 µl of TOP10 E. coli with 4 µl of DNA in TE for all 22 parts, plated transformed bacteria and incubated overnight at 37°C.

05/20/08
 * We used LB medium previously prepared, with the suitable antibiotic added.
 * After overnight incubation, the following 14 plates showed colonies:


 * While the following plates did not:


 * Plate containing BBa_J23100 showed red colonies, as we expected: this part is inserted into plasmid BBa_J61002 which places the RFP downstream of the inserted part, which is a constitutive promoter.


 * We picked up one colony from every working plate to grow 5 ml cultures of transformed bacteria overnight.


 * We re-transformed 60 µl of TOP10 with the remaining 6 µl of DNA in TE for BBa_I14032, BBa_R0051, BBa_I15008, BBa_I15009, BBa_I15010, BBa_C0161, BBa_C0078, BBa_C0179.


 * We plated transformed bacteria and incubated them at 37°C overnight.


 * We ordered primers VF2 and VR. We also ordered new TOP10 stocks to Invitrogen, because our stock was about to run out.

05/21/08
 * Only BBa_I15009 and BBa_C0078 plates showed colonies and for the remaining 6 plates there were no colonies again.


 * We picked up one colony from BBa_I15009 and BBa_C0078 plates to grow 5 ml cultures of transformed bacteria overnight.


 * We re-cut paper spots for BBa_R0051, BBa_I14032, BBa_I15008, BBa_I15010, BBa_C0161, BBa_C0179 and resuspended them again in 10 µl of warmed TE buffer.


 * We repeated the transformation for these 6 parts using 4 µl of DNA in TE.


 * We plated transformed bacteria and incubated them at 37°C overnight.


 * We prepared 14 glycerol stocks taking 800 µl from 5 ml cultures containing:


 * We performed plasmid purification for these 14 parts.

05/22/08
 * For the third time, there were no colonies in the 6 plates.


 * We contacted IGEM Headquarters to explain our problem for these 6 parts. We thank Meagan Lizarazo for her kind attention! IGEM Headquarters will send us the 6 parts.


 * We tried to transform these 6 parts for the last time, using the remaining 6 µl of DNA in TE. We used our last 6 TOP10 stocks.


 * We prepared 2 glycerol stocks taking 800 µl from 5 ml cultures containing BBa_I15009 and BBa_C0078.


 * We performed plasmid purification for these 2 parts.


 * We had QIAGEN mini kit at our disposal, instead of QIAGEN miniprep kit for plasmid purification. We noticed that QIAGEN mini kit performed a low yield extraction (40-80ng/µl instead of 200-500ng/µl normally yielded by miniprep kit): quantified plasmid concentrations measured with NanoDrop spectrophotometer were very low.


 * For this reason we decided to re-perform plasmid extraction with a higher culture volume: 9 ml instead of 5 ml. Anyway, we are waiting to receive QIAGEN miniprep kit in order to perform more efficient plasmid purifications.


 * We took 15 µl from all our 16 glycerol stocks and infected 9 ml LB + suitable antibiotic for each part we had. We incubated the 9 ml cultures at 37°C overnight.

05/23/08
 * For the fourth time, there were no colonies in the 6 plates. We decided to wait for the 6 paper spots from IGEM Headquarters, instead of performing another cut/transformation.


 * We prepared 14 glycerol stocks taking 800 µl from our 10 ml cultures.


 * We performed plasmid purification for all cultures.


 * Quantification of plasmid concentration showed that 9 ml cultures yielded higher amounts of plasmid than using 5 ml cultures (100-150ng/µl instead of 40-80ng/µl).


 * Center for Tissue Engineering (CIT) meeting: we explained our first results to CIT members.