Team:UNIPV-Pavia/Notebook/Week3Aug
From 2009.igem.org
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Week from August 17th, to August 23rd, 2009
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Next Week
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August, 17th
- This week we planned to perform a gel run for all the constructs of the ethanol producing operon in order to check for contaminant bands. Then, we planned to ligate a promoter upstream of the ethanol producing operon and to build up measurement systems for A11 (lactose sensor) and A12 (aTc sensor).
- We inoculated 8 ul of:
- B1-13
- B2-5
- B3-5
- B4-2
- B5-3
- B6-3
- E0240
- A11-2
- glycerol stocks in 5 ml of LB + suitable antibiotic.
- We incubated these cultures overnight (37°C, 220 rpm).
Preparation of experiment with Tecan F200
- We dissolved 22 mg of lactose in 5 ml of LB + Kan in order to have 4.5% lactose.
- We prepared lactose assay kit for testing.
Experiment with Tecan F200
August, 18th
- Glycerol stock for E0240, A11-2, B1, B2, B3 and B4 in order to have a backup.
- Miniprep for:
- B1-13
- B2-5
- B3-5
- B4-2
- B5-3
- B6-3
- E0240
- A11-2
- Digestion with EcoRI for:
- B1-13
- B2-5
- B3-5
- B4-2
- B5-3
- B6-3
- Digestion with EcoRI-PstI for:
- B1-13
- B2-5
- B3-5
- B4-2
- B5-3
- B6-3
- Digestion with PstI for:
- B1-13
- B2-5
- B3-5
- B4-2
- B5-3
- B6-3
- Electrophoresis for the digested plasmids.
- Gel results:
- B1 - ok
- B2 - ok
- B3 - two extra-bands (consistent with previous gels)
- B4 - ok
- B5 - two extra bands (consistent with previous gels)
- B6 - two extra bands (consistent with previous gels)
- We decided to try to ligate a promoter upstream of B5 and B6 anyway.
- We inoculated 8 ul of:
- A4(X2)
- A12
- glycerol stocks.
- We incubated these inocula at 37°C, 220 rpm overnight.
August, 19th
- Digestion for:
- E0240(X-P)
- A11-2(S-P)
- B5(E-X)
- B6(E-X)
- Miniprep for:
- A4(X2)
- A12
- Digestion for:
- A4(E-S)(X2)
- A12(S-P)
- Precipitation with sodium acetate for:
- A11-2(S-P)
- B5(E-X)
- B6(E-X)
- A4(E-S)(X2)
- A12(S-P)
- Gel run/cut/purification for E0240.
- Ligations:
- B7 = A4(E-S) + B5(E-X) in pSB1AK3
- B8 = A4(E-S) + B6(E-X) in pSB1AK3
- A14 = A11(S-P) + E0240(X-P) in pSB1A2
- A16 = A12(S-P) + E0240(X-P) in pSB1A2
Experiment with Tecan F200
August, 20th
- We resuspended pSB3K3 from 2009 Registry Distribution. The plasmid with ccdB was not consistent, so we resuspended a consistent brick (of ~700bp length, suggested in the Registry "Help" page: Kit Plate 2, well 15L, I714891 brick) contained in pSB3K3.
- We transformed the overnight ligations and I714891 in TOP10 and plated transformed bacteria on LB agar plates + Amp (A14 and A16) or + Kan (pSB3K3, B7 and B8). We incubated the plates at 37°C overnight.
- We sent these samples (stored at -20°C) to BMR Genomics for sequencing:
- A15-1
- A15-3
- A11-2
- A11-3
- B5-3
- B6-3
- B3-5 (again, because we wanted to check for contaminants in our native stock)
August, 21st
- Colony PCR for A14 (5 colonies) and A16 (5 colonies) plates. Colonies were inoculated in 1 ml of LB + Amp and let grow waiting for the end of the reaction.
- Electrophoresis for PCR results.
- Gel results (picture not taken, sorry...):
- A14 - 3rd colony seems the most pure and it had the expected length for ligated plasmid.
- A16 - reaction worked only on A16-1, but it was negative.
- We decided to keep A14-3 (positive at PCR) and A16-4 (randomly chosen) for digestion screening: we prepared a glycerol stock for them and re-filled the remaining 250 ul of bacteria with 4 ml of LB + Amp.
- We incubated these cultures at 37°C, 220 rpm overnight.
- We picked 2 colonies from B7, 5 colonies from B8 and 1 colony from I714891 plates. We inoculated them in 1 ml of LB + Kan and incubated them at 37°C, 220 rpm for 5 hours and 1/2. Then we prepared glycerol stocks and re-filled the remaining 250 ul with 4 ml of LB + Kan.
- NOTE: we decided not to perform PCR on B7 and B8 plates because of the large size of positive inserts.
- We incubated the re-filled cultures at 37°C, 220 rpm overnight.
Experiment with Tecan F200
August, 22nd
- Pellet preparation for the 10 overnight cultures, ready to be miniprepped next week! Pellets were stored at -20°C.
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Next Week
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