Team:UNIPV-Pavia/Notebook/Week1

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Notebook

Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7
Week 8	Week 9	Week 10	Week 11	Week 12	Week 13	Week 14
Week 15	Week 16	Week 17	Week 18	Week 19	Week 20	Week 21
Week 22	Week 23	Week 24

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:

BBa_I14032	BBa_R0079	BBa_R0062	BBa_R0040
BBa_R0082	BBa_R0051	BBa_J23100	BBa_C0161
BBa_C0062	BBa_C0078	BBa_C0179	BBa_C0051
BBa_C0012	BBa_C0040	BBa_I15010	BBa_I15008
BBa_I15009	BBa_E0040	BBa_E1010	BBa_B0030
BBa_B1006	BBa_E0240

Registry of Standard Parts 2008 in our lab

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.

Our 22 parts at 37°C

We used LB medium previously prepared, with the suitable antibiotic added.

05/20/08

After overnight incubation, the following 14 plates showed colonies:

BBa_R0079	BBa_R0062	BBa_R0040	BBa_R0082
BBa_J23100	BBa_C0062	BBa_C0051	BBa_C0012
BBa_C0040	BBa_E0040	BBa_E1010	BBa_B0030
BBa_B1006	BBa_E0240

BBa_E0240

While the following plates did not:

BBa_I14032	BBa_R0051	BBa_C0161	BBa_C0078
BBa_C0179	BBa_I15008	BBa_I15009	BBa_I15010

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:

BBa_R0079	BBa_R0062	BBa_R0040	BBa_R0082
BBa_J23100	BBa_C0062	BBa_C0051	BBa_C0012
BBa_C0040	BBa_E0040	BBa_E1010	BBa_B0030
BBa_B1006	BBa_E0240

Two glycerol stocks: BBa_J23100 red culture and BBa_B0030 normal color culture

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.