Team:UNIPV-Pavia/Notebook/Week2

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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 2: 05/26/08 - 05/31/08

05/26/08

  • Team meeting at Biomedical Informatics Lab to talk about UNIPV-Pavia team presentation at Europe Teachers' Workshop in Paris and to make our first wiki updating.
  • We received restriction enzymes and Agarose Gel DNA Extraction Kit from Roche. Now we are ready to cut;)



05/27/08

  • We performed plasmid digestion for these 9 parts (1 µg):
BBa_C0078 (X-P) BBa_E0040 (X-P) BBa_E1010 (X-P) BBa_C0051 (X-P)
BBa_B0030 (X-P) BBa_B0030 (E-X) BBa_B0030 (S-P) BBa_E0240 (E-X)
BBa_J23100 (E-S)
  • We had to insulate excided fragments for:
BBa_C0078 (X-P) BBa_E0040 (X-P) BBa_E1010 (X-P) BBa_C0051 (X-P)
BBa_B0030 (X-P) BBa_J23100 (E-S)
  • While we had to insulate opened plasmids for:
BBa_B0030 (E-X) BBa_B0030 (S-P) BBa_E0240 (E-X)
  • We ran two different gels for these two groups, because excided fragments are smaller than opened plasmids.
  • Unfortunately, BBa_B0030 (X-P) and BBa_J23100 (E-S) fragments had smearing appearance because they were smaller than other excided fragments and ran too fast.
  • We performed gel extraction for all the 9 parts, but we decided to repeat cutting/run/extraction process for BBa_B0030 (X-P) and BBa_J23100 (E-S) in the next days, to perform a more efficient extraction for these 2 parts.
  • We took 15 µl from BBa_B0030 and BBa_J23100 glycerol stocks and infected 9 ml LB + Amp for each part. We incubated the 9 ml cultures at 37°C overnight. (We already had at our disposal extracted plasmids for these 2 parts, but we performed plasmid extraction anyway to have more).



05/28/08

  • We performed plasmid digestion for these 4 parts (about 5.5 µg for BBa_C0062; the whole quantity for the others):
BBa_C0062 (X-P) BBa_C0012 (X-P) BBa_C0040 (X-P) BBa_I15009 (X-P)
  • NOTES: to avoid smearing fragments during electrophoresis, we planned experiments in which only excided fragments of the same order of magnitude are ran in the same gel. So, we decided to run these four parts on May 28 (the smallest part is 660 bp and the largest one is 1128 bp). The remaining parts will be digested/gel extracted on May 29 (the smallest part is 39 bp and the largest one is 157 bp).
  • We loaded and ran a gel to separate these 4 excided fragments from the rest of their plasmid.
  • We performed gel extraction.
  • We expected to find the 9 ml culture for BBa_J23100 red, but it was not! So we performed miniprep only for BBa_B0030 9 ml culture, after taking 800 µl from 9 ml BBa_B0030 culture to prepare a new glycerol stock.
  • Unfortunately DNA pellet washing failed...Unlucky day for minipreps!
  • We took 15 µl again from BBa_B0030 and BBa_J23100 glycerol stocks and infected 9 ml LB + Amp for each part. We incubated the 9 ml cultures at 37°C overnight.



05/29/08

  • We received VF2 and VR primers.
  • We received the 6 parts we had requested to IGEM 2008 Headquarters! (5 µl DNA + glycerol for every part) IGEM HQ also sent us a new punch tool:) Thank you very much!
  • 9 ml cultures were grown correctly and BBa_J23100 culture was red. So we could perform miniprep for these 2 parts.
Red pellet for BBa_J23100 culture after a 15 min 4°C 6000 x g centrifuging
  • Unfortunately, plasmids extracted from BBa_B0030 culture were not pure: quantification at spectrophotometer showed an extra peak at 280 nm, which corresponds to protein contaminations.
  • We will repeat miniprep for BBa_B0030 next week, hoping to be luckier than this week...and hoping that QIAGEN miniprep kit will arrive soon!
  • We performed plasmid digestion for these 5 parts:
BBa_R0079 (X-P) BBa_R0040 (X-P) BBa_R0082 (X-P) BBa_R0062 (X-P) BBa_J23100 (E-S)
  • We loaded and ran a gel to separate these 5 excided fragments from the rest of their plasmid.
  • We performed gel extraction.
  • Wiki updating



05/30/08

  • Wiki updating
  • We received TOP10 stocks in the afternoon: next week we will transform the 6 parts we received on May 29.



05/31/08

  • Europe Teachers' Workshop in Paris


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