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Team:UNIPV-Pavia/Notebook/Week2Aug
From 2009.igem.org
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*M9 supplemented medium preparation (with glycerol). This first attempt showed precipitations...we will try tomorrow to dissolve MgSO4 and CaCl2 in ddH2O very slowly. | *M9 supplemented medium preparation (with glycerol). This first attempt showed precipitations...we will try tomorrow to dissolve MgSO4 and CaCl2 in ddH2O very slowly. | ||
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| + | ''Preparation of experiment with Tecan F200'' | ||
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| + | *We inoculated 10 ul of A2 glycerol stock and a single colony of B0033 from its native plate in 5 ml of LB + Amp. | ||
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| + | *We incubated the cultures overnight (37°C, 220 rpm). | ||
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Revision as of 13:49, 15 August 2009
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Week from August 10th, to August 16th, 2009
Previous Week
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Next Week
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August, 10th
- Digestion for:
| B1-13(E-S)(X2) | B2-5(E-S)(X2) | B3-5(E-X)(X2) |
| B4-2(E-X)(X2) |
- Gel run for all of them (only 1 ul for B1-13(E-S)(X2) in order to check the length)
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- Band cut/purification for:
| B2-5(E-S)(X2) | B3-5(E-X)(X2) | B4-2(E-X)(X2) |
- Ethanol precipitation with sodium acetate for B1-13(E-S)(X2).
- Ligation:
- B5 = B1(E-S) + B4(E-X) in pSB1AK3
- B6 = B2(E-S) + B3(E-X) in pSB1AK3
- We incubated the ligation reactions at 16°C overnight.
August, 11st
- We transformed the overnight ligations of B5 and B6. We plated the transformed bacteria on LB agar plates + Kan and incubated the plates at 37°C overnight.
- Digestion for:
| BOL1(E-S) | R0010(E-X) | F2620MIT1(E-S) |
| K112808(E-X) |
- Gel run/cut/purification for all of them. All the bands were present at the right size!
- Ligation (20 ul final volume) for:
- A11 = BOL1(E-S) + R0011(E-X) in pSB1A2 (we call it A11 again, because the previous A11 had a deletion ad we threw it away)
- A15 = F2620MIT1(E-S) + K112808(E-X) in pSB1A2
- We incubated the ligations at 16°C overnight.
- M9 supplemented medium preparation (with glycerol). This first attempt showed precipitations...we will try tomorrow to dissolve MgSO4 and CaCl2 in ddH2O very slowly.
Preparation of experiment with Tecan F200
- We inoculated 10 ul of A2 glycerol stock and a single colony of B0033 from its native plate in 5 ml of LB + Amp.
- We incubated the cultures overnight (37°C, 220 rpm).
August, 12nd
- We transformed 1 ul of A11 and A15 ligations in TOP10. We plated on LB + Amp agar plates and incubated them in the morning.
- After about 9 hours the plates showed many colonies! we picked three single colonies from each plate and infected 5 ml of LB + Amp. We incubated these inocula at 37°C, 220 rpm overnight to grow up cultures to screen.
- We picked 7 colonies from B5 and B6 overnight plates and infected 1 ml of LB + Kan. We incubated these 14 inocula at 37°C, 220 rpm for 6 hours and then we prepared glycerol stocks.
- We re-filled the remaining 250 ul with 5 ml of LB + Kan to grow overnight cultures to screen.
- We repeated M9 supplemented medium preparation and we finally had it without precipitations!
August, 13rd
August, 14th
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Previous Week
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Next Week
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