Team:UNIPV-Pavia/Notebook/Week3

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Revision as of 17:39, 7 June 2008

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Notebook

Week 3: 06/03/08 - 06/06/08

06/03/08

• We transformed 60 µl of TOP10 with 2 µl of the 6 parts (DNA + glycerol) we received from IGEM HQ:

• NOTES: We noticed that IGEM 2007 teams which used luxI or lasR parts choosed BBa_C0061 instead of BBa_C0161 (for luxI) and BBa_C0079 instead of BBa_C0179 (for lasR). So, we decided in addition to amplify BBa_C0061 and BBa_C0079; we shall see later which luxI and lasR to choose for our project.

• So, we cut paper spots for BBa_C0061 and BBa_C0079 and resuspended them in 10 µl of warmed TE buffer.

• We transformed these 2 parts using 4 µl of DNA in TE.

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

06/04/08

• After overnight incubation, the following 5 plates showed colonies:

BBa_C0061 BBa_R0051 BBa_I14032 BBa_I15008 BBa_I15010

BBa_R0051 plate: very high yield from IGEM HQ DNA + glycerol

BBa_C0061 plate: medium yield from paper spot

• while the following 3 plates did not:

• We repeated the transformation for BBa_C0079, BBa_C0179 and C0161. We used 6 µl of DNA in TE for BBa_C0079, while we used 3 µl of DNA + glycerol for BBa_C0179 and BBa_C0161.

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

• While we were preparing our 5 ml cultures for the 5 working plates, we noticed that LB + Amp was contaminated! We decided to prepare a big quantity of LB + Amp and also of LB + Kan: we prepared 0.5 l LB + Amp and 0.5 l LB + Kan for liquid cultures; 0.5 l LB + Amp and 0.5 l LB + Kan for plates.

A lot of just prepared LB plates

• We picked up one colony from BBa_C0061, BBa_R0051, BBa_I14032, I15008 and I15010 plates to grow 5 ml cultures of transformed bacteria overnight.

• We also infected 5 ml of LB + Amp with 15 µl of BBa_B0030 glycerol stock. We incubated the 5 ml cultures overnight at 37°C.

• We received QIAGEN QIAprep Spin Miniprep Kit!!! We will inaugurate it tomorrow on these 5 ml cultures;)

06/05/08

• We received Euroclone Antartic Phosphatase: we will use it in the afternoon before ligation reaction!

• We prepared 6 glycerol stocks taking 800 µl from 5 ml cultures containing:

• We performed miniprep for these 6 parts with our new splendid kit;) Plasmid quantification confirmed a higher yield than our previous QIAGEN kit.

• We performed plasmid digestion for these parts (20 of 30 µl).

• We had to insulate excided fragments for:

• While we had to insulate opened plasmids for:

• Due to the different dimension of the DNA we had to insulate, we ran 3 different gels:
  • one for BBa_R0051 (S-P)
  • one for BBa_I14032 (X-P) and BBa_B0030 (X-P)
  • one for BBa_C0061 (X-P), BBa_I15008 (X-P) and BBa_I15010 (X-P).

• We could see and cut all the bands we wanted, except for BBa_I14032, for which we couldn't see any band.

• We performed gel extraction for:

• All the 3 overnight plates worked (low yield for all).

• We picked up one colony from BBa_C0161, BBa_C0179 and BBa_C0079 plates to grow 5 ml cultures of transformed bacteria overnight. We did the same thing for BBa_I14032 6/3/08 plate; we didn't use glycerol stock for BBa_I14032 because we thought there was something wrong with the colony we picked on 6/4/08.

• We calculated the amount of vector/insert for ligation reaction to have a molarity ratio of 2:1 (insert:vector).

• We performed ligation reaction for (vectors are embolded):

1. BBa_J23100-BBa_E0240        (Pcon(E-S)-assGFP(E-X))
2. BBa_J23100-BBa_B0030        (Pcon(E-S)-RBS(E-X))
3. BBa_R0051-BBa_B0030        (Plam(S-P)-RBS(X-P))
4. BBa_B0030-BBa_E0040        (RBS(S-P)-GFP(X-P))
5. BBa_B0030-BBa_C0051        (RBS(S-P)-cI(X-P))
6. BBa_B0030-BBa_E1010        (RBS(S-P)-RFP(X-P))
7. BBa_B0030-BBa_C0061        (RBS(S-P)-luxI_LVA(X-P))
8. BBa_B0030-BBa_C0078        (RBS(S-P)-lasI(X-P))

• We incubated ligation overnight at 16°C.

06/06/08

• We took 800 µl of BBa_C0161, BBa_C0179, BBa_C0079 and BBa_I14032 overnight cultures to prepare glycerol stocks.

• We prepared 4 glycerol stocks taking 800 µl from 5 ml cultures containing:

• Miniprep for BBa_C0161, BBa_C0179, BBa_C0079 and BBa_I14032.

• We performed plasmid digestion for these 4 parts (20 of 30 µl).

• We ran two different gels:
  • one for BBa_C0161 (X-P), BBa_C0179 (X-P) and BBa_C0079 (X-P)
  • one for BBa_I14032 (X-P), smaller than the other 3 parts

• For the second time we couldn't see any band for BBa_I14032.

• We performed gel extraction for:

BBa_C0161 (X-P) BBa_C0179 (X-P) BBa_C0079 (X-P)

Mattia cutting BBa_C0161 (X-P), BBa_C0179 (X-P) and BBa_C0079 (X-P) gel

• We decided to sequence BBa_I14032 to check if the sequence is correct. Unfortunately we had not enough plasmid and so we picked another colony from 6/3/08 plate, infected 9 ml LB + Kan and incubated it overnight at 37°C.

• We transformed the whole ligation volume (30 µl) for all the 8 overnight ligations.

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

06/07/08

• Only BBa_J23100-BBa_B0030 (Pcon(E-S)-RBS(E-X)) plate showed colonies. We put it at 4°C.

• We took 800 µl of BBa_I14032 overnight culture to prepare a glycerol stock.

• Miniprep for BBa_I14032 9 ml culture to prepare the sample for sequencing.

• We sent BBa_I14032 sample and VF2 primer to Primm for sequencing.

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