Week from July 6th, to July 12nd, 2009

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July, 6th

• Digestion for:

• Gel run/cut and band purification.

• The purified DNA gave a good result at Nanodrop, but we decided to perform the entire work on BOL1 again because we were not sure that we had extracted a pure band.

• We stored R0011(E-X) DNA at -20°C.

• We infected 5 ml of LB + Amp with 8 ul of BOL1 glycerol stock to grow two identical overnight cultures (37°C, 220 rpm).

Preparation of experiment with Tecan F200

• We picked a colony of B0030 plate (stored at +4°C) and infected 5 ml of LB + Amp.

• We also infected 5 ml of LB + Amp with 10 ul of A1 and J23100 glycerol stocks.

• We incubated the inocula overnight at 37°C, 220 rpm.

July, 7th

• Miniprep for BOL1 (X2).

• Digestion for BOL1(E-S)(X2).

• Gel run/cut/purification: none of the two BOL1 samples gave a good results in DNA quantification...we decided to cut the miniprepped plasmids again, but this time through an overnight digestion (both plasmids E-S).

• In case of failure, we also infected 5 ml of LB + Amp with 8 ul of BOL1 glycerol stock to grow an overnight culture at 37°C, 220 rpm.

• We prepared 0.5 l of LB + Amp.

• We received sequencing results for:
  • T9002 - long part, but SEQUENCE CONFIRMED! while MIT results were "INCONSISTENT".
  • A10 - results showed a good chromatogram, but K117000 had not been correctly ligated. We will repeat this ligation one of the next weeks.

Preparation of experiment with Tecan F200

• We diluted 1:100 the overnight cultures of A1, B0030 and J23100.

• We incubated the diluted cultures for 5 hours (37°C, 220 rpm).

Experiment with Tecan F200

• Description
• Purpose:
• Materials & Methods
• Protocol
• Results

Preparation of tomorrow's experiment with Tecan F200

• We picked a colony from B0030 native plate (stored ad +4°C) and infected 5 ml of LB + Amp. We incubated the inoculum at 37°C, 220 rpm overnight.

July, 8th

• Gel run/cut/band purification for BOL1(E-S) overnight digestion. Results: gel showed that the enzymes had cut very well and this time also the purification gave a good quantification at Nanodrop.

• We took R0011(E-X) stored ad -20°C and we were ready to ligate;)

• Ligation:
  • A11: BOL1(E-S) + R0011(E-X) in pSB1A2

• We incubated the ligation overnight at 16°C.

Preparation of experiment with Tecan F200

• We diluted 1:100 the overnight culture of B0030.

Experiment with Tecan F200 (continues the next day)

• Description
• Purpose:
• Materials & Methods
• Protocol
• Results

July, 9th

• We resuspended F2620 BioBrick from iGEM 2009 plates.

• We transformed about 40 pg of the overnight ligation and 1 ul of the resuspended F2620 BioBrick. We plated transformed bacteria and incubated the two plates overnight at 37°C.

End of experiment with Tecan F200 (last measurement)

July, 10th

• A11 and F2620 overnight plates showed colonies!

• Colony PCR for 6 colonies of A11 plate: the picked colonies were inoculated in 1 ml of LB + Amp and incubated at 37°C, 220 rpm waiting for PCR results.

• We also picked a colony from F2620 to infect 1 ml of LB + Amp. We incubated this inoculum with the others.

Colony PCR for A11 plate: all the colonies showed the correct plasmid. We kept the 1st, 3rd and 6th colonies.

• Gel results: all the picked colonies showed the expected amplicon of a correctly ligated plasmid! We decided to prepare a glycerol stock for:
  • A11-1
  • A11-3
  • A11-6

• Next week, only A11-1 will be used, but the other colonies will be used in case of failure.

• We also prepared a glycerol stock for F2620 culture.

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