Team:UNIPV-Pavia/Notebook/Week1

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*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.
*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.
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[[Image:pv_22plates.jpg|thumb|300px|left|Our 22 parts at 37°C]]
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*We used LB medium previously prepared, with the suitable antibiotic added.
*We used LB medium previously prepared, with the suitable antibiotic added.
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Revision as of 16:34, 29 May 2008


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Notebook



Week 1 Week 2



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 10 ml LB + suitable antibiotic for each part we had. We incubated the 10 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).
  • Tissue Engineering Center (CIT) meeting: we explained our first results to CIT members.
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