Team:UNIPV-Pavia/Notebook/Week9

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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 8: 07/14/08 - 07/17/08

07/14/08

  • We transformed 1 µl of the 6 ligations stored at -20°C. We plated transformed bacteria and incubated plates at 37°C overnight.
  • Plasmid digestion for:
BBa_R0079 (E-X) BBa_B0030-BBa_C0078-BBa_B1006 (E-S)
  • Gel run/cut/gel extraction for the 2 parts.
  • Ligation: BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079.
  • We incubated ligation at 16°C overnight.



07/15/08

  • We transformed 1 µl of BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 ligation and plated transformed bacteria. We incubated the plate at 37°C overnight.
  • All the 6 overnight plates showed colonies. We performed colony PCR for (we decided to pick up more colonies for the ligations with long inserts):
  1. BBa_J23100-BBa_B0030 (S-P) -BBa_C0012 (standard ligation) - 10 colonies
  2. pSB1AK3-BBa_J23100-BBa_B0030-BBa_C0012 (double ligation) - 7 colonies
  3. BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040 - 3 colonies
  4. BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 - 3 colonies
  5. BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006 - 10 colonies
  6. BBa_B0030-BBa_I15009-BBa_B1006 - 3 colonies
Picked up colonies in LB + Amp
File:Pv pcr 9d 9s 26 28 30 31.jpg
  • Gel results:
  1. BBa_J23100-BBa_B0030-BBa_C0012 (standard ligation) - 9th colony
  2. pSB1AK3-BBa_J23100-BBa_B0030-BBa_C0012 (double ligation) - no true positives
  3. BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040 - no true positives
  4. BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 - 3rd colony
  5. BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006 - 7th colony
  6. BBa_B0030-BBa_I15009-BBa_B1006 - 1st colony
  • Comments:
    • double ligation apparently did not work, but this time we had our true positive colonies for BBa_J23100-BBa_B0030-BBa_C0012 with a standard ligation.
    • BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040 did not show colonies with ligated plasmid...Maybe we picked up too too few colonies (in addition, we probably made a mistake with the 2nd colony which was not dipped in PCR tube...). We will repeat colony PCR on this ligation soon.
  • We infected 9 ml LB + Amp with these glycerol stocks:
BBa_R0082 BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006
  • We grew the 4 chosen colonies and the 2 glycerol stocks colonies overnight.



07/16/08

  • BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 plate showed a carpet...We decided to streak the plate to grow a "single colonies" plate overnight. We also decided to perform "colony" PCR (we did not use colonies, but little streaks) to check if there were ligated plasmids.
  • So, we performed colony PCR for BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 and we repeated colony PCR for BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040.
Pv pcr 25 28 .jpg
  • Gel results:
    • All the 8 BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 streaks contained ligated plasmid! but gel run showed an extra band that could correspond to not ligated plasmid...We decided to perform colony PCR on single colonies plate. We also decided to grow a 9 ml culture overnight with the 4th colony (in which the extra band is very weak) to have a backup of this result.
    • BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040 showed some pure true positive colonies; we chose the 2nd colony to grow a 9 ml culture overnight.
  • Glycerol stocks for:
BBa_R0082 BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006
BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (3) BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006 (7)
BBa_B0030-BBa_I15009-BBa_B1006 (1) BBa_J23100-BBa_B0030-BBa_C0012 (9)
  • Miniprep for these 6 parts.
  • Plasmid digestion for:
BBa_R0082 (E-X) BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (X-P)
BBa_J23100-BBa_B0030-BBa_C0012 (9) (E-S) BBa_B0030-BBa_I15009-BBa_B1006 (1) (E-S)
BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (3) (X-P) BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006 (7) (S-P)
  • Gel run/cut/gel extraction for these 6 parts.



07/17/08

  • Glycerol stocks for BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 (4) and BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040 (2).
  • We put BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 single colonies plate at +4°C.
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