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Week from July 27th, to August 2nd, 2009

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July, 27th

• Screening for the 11 miniprepped DNA samples for B1: digestion S-P for all.

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• Gel results:
  • Samples 1, 4, 10, 11, 14, 19 and 20 showed an extra band for the non ligated plasmid;
  • Samples 2, 9, 13 and 18 were pure! we decided to keep B1-13 (lane 8) to perform future ligations.

• NOTE: we had a pure sample for B1 (i.e. B1-13)and three almost-pure sample for B2. Anyway, we decided to perform ligation reactions for these samples and the extra band of B2 will be eliminated during gel cut/purification. WE DECIDED TO KEEP B2-5. Next weeks we will think about purifying B2-5 itself.

• We transformed 20 pg of B1-13 purified DNA (stored at -20°C) in TOP10 in order to prepare a glycerol stock for this construct. We incubated the plate at 37°C overnight.

Preparation of experiment with Tecan F200

• We infected 5 ml of LB + Amp with 10 ul of A14pg, A8pg and A9pg glycerol stocks.

• We also infected 5 ml of LB + Amp with a single colony taken from B0030 native plate (stored at +4°C).

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

July, 28th

• We streaked LB agar plates + suitable antibiotic with iGEM stabs:

K116001	K116002	K112405
P0412	I746902	I746903
K101017	F2620MIT1	F2620MIT2

• We incubated these "single colonies" plates at 37°C overnight.

• We picked a single colony from B1-13 plate to infect 1 ml of LB + Amp and incubated this inoculum for 5 hours and 1/2.

• We prepared a glycerol stock for B1-13.

• We aliquoted the remaining 250 ul of B1-13 bacterial culture in two different falcon tubes and re-filled them with 5 ml of LB + Amp.

• We also infected 5 ml of LB + Amp with 10 ul of B0015(X2) and B2-5(X2) glycerol stocks.

• We incubated these six cultures at 37°C, 220 rpm overnight.

• We received sequencing results for:
  • A12-2: sequence ok!
  • A12-3: sequence ok!

Preparation of experiment with Tecan F200

• We diluted 1:1000 the overnight cultures of A14pg, A8pg, A9pg and B0030.

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

• After 5 hours, we adjusted the OD600 at 0.025.

Experiment with Tecan F200

• Download Protocol

July, 29th

• Miniprep for:
  • B1-13
  • B1-13bis
  • B2-5
  • B2-5bis
  • B0015
  • B0015bis

• Digestion:
  • B1-13(E-S)
  • B1-13bis(E-S)
  • B2-5(E-S)
  • B2-5bis(E-S)
  • B0015(E-X)
  • B0015bis(E-X) - 500ng

• Gel run/cul/purification for:
  • B1-13(E-S) - ONLY FOR CHECK, NOT PURIFIED
  • B1-13bis(E-S) - ONLY FOR CHECK, NOT PURIFIED
  • B2-5(E-S)
  • B2-5bis(E-S)
  • B0015(E-X)
  • B0015bis(E-X)

• Precipitation with sodium acetate for:
  • B1-13(E-S)
  • B1-13bis(E-S)

All the bands were in the right place!

• We had good yields for all, except from B0015bis(E-X). We will use the other vector (i.e. B0015(E-X)) for ligation.

• Ligation:
  • B3 = B1(E-S) + B0015(E-X) in pSB1AK3 (50 ng of vector)
  • B4 = B2(E-S) + B0015(E-X) in pSB1AK3 (26 ng of vector)
• keeping the sample that gave the higher yield.

• We incubated the ligations at 16°C overnight.

• All the streaked plates showed single colonies! we prepared an inoculum for all of them picking a single colony and infecting 1 ml of LB + suitable antibiotic. We incubated these inocula for 5 hours and 1/2.

• Then, we prepared a glycerol stock for each of them and re-filled the remaining 250 ul of bacterial culture with 3 ml of LB + suitable antibiotic.

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

July, 30th

• We transformed B3 (50 pg) and B4 (20 pg) ligations. We incubated the plates at 37°C overnight.

• Miniprep for the overnight cultures of:

K116001	K116002	K112405
P0412	I746902	I746903
K101017	F2620MIT1	F2620MIT2

• We sent purified DNA to BMR Genomics for sequencing (VF2 and VR for all of them, except from K112405, whose plasmid does not have VF2 annealing site).

• We also sent purified DNA of B1-13 and B2-5 (stored at -20°C) to BMR Genomics for sequencing.

• We received sequencing results for A11: lacI sequence showed a deletion...we will repeat A11 ligation from BOL1 and R0011 parts.

Preparation of experiment with Tecan F200 (for the following day!)

• We inoculated 10 ul of J23100 and J23100-E0240 parts form UNIPV iGEM 2008 stocks.

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

July, 31st

• B3 and B4 plates showed colonies! We picked 7 colonies for each plate and infected 1 ml of LB + Kan. We incubated these 14 inocula for 5 hours and 1/2. Then we prepared glycerol stocks and re-filled the remaining 250 ul of bacterial culture with 5 ml of LB + Kan.

• We incubated these cultures at 37°C, 220 rpm overnight.

Preparation of experiment with Tecan F200

• We diluted 1:100 J23100 and J23100-E0240 parts form UNIPV iGEM 2008 stocks overnight cultures.

• We incubated these two cultures at 37°C, 220 rpm for about 3 hours.

August, 1st

• Miniprep for the 7 colonies of B3 and for the 7 colonies of B4. We stored purified DNA at -20°C and next Monday we will perform screening for these 14 samples.

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