Team:UNIPV-Pavia/Notebook/Week2Jul

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

July, 6th

 * We planned to dedicate this week to the assembly of an IPTG/lactose->PoPS device in order to have a lactose sensor for our project, but also a useful inducible system for general purpose applications.


 * We already had R0011 and BOL1 purified plasmids stored at -20°C. 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.

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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


 * Download Protocol

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.

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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.

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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.

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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.




 * 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.


 * We contacted iGEM HQ to request these BioBricks:

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