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Team:Groningen/Notebook/21 August 2009
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PCR on pBAD plasmid with primers F2 rev has worked. cut out and isolated 11 ug/ul | PCR on pBAD plasmid with primers F2 rev has worked. cut out and isolated 11 ug/ul | ||
Used in 3 PCRs, (pcr1)F1 mut1rc, (pcr2)Rev mut2rc and F1 Rev. | Used in 3 PCRs, (pcr1)F1 mut1rc, (pcr2)Rev mut2rc and F1 Rev. | ||
| + | |||
| + | PCR worked. Isolated and pcr to get the final construct. | ||
===Metal Accumulation=== | ===Metal Accumulation=== | ||
| + | *'''PCR to amplify fMT, SmtA with pre-RBS''' | ||
| + | **Redo PCR from yesterday as the PCR didnt proceed (hot start was manually) | ||
| + | **Run on gel and excise bands. | ||
| + | |||
| + | *'''Ligate pSB1AC3 with MymT, fMT and SmtA''' | ||
| + | **Do restriction of the vector and the inserts with EcoRI and SpeI. | ||
| + | ** Do PCR clean-up kit to purify from the restriction enzymes. | ||
| + | ** Check concentration and ligate o/n @ 4°C 100ng in a 3x overexcess of insert. | ||
===Vectors=== | ===Vectors=== | ||
Latest revision as of 16:44, 21 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
Ligation
(1:?)
- 2 uL Ligase buffer
- 1 ul T4 Ligase
- 2 uL plasmid BBa_J61002 digested with EcoRI and SpeI
- 1 uL insert oligo's with pMetal+RBS EcoRI/SpeI ends
Incubate:
- 25°C 60min.
- kept on ice for 10min.
Tranformation
- add 10uL of the pSB1AC3-GVP ligation product to 50uL competent E.coli TOP10 cells.
Incubate:
- 30 min @ ice
- 90 sec 42°C
- 2 min @ ice
- add 800uL LB-medium
- incubate for 1 h at 37°C
- plate on LB-amp100 plates
Over Night Cultures
Transporters
PCR on pBAD plasmid with primers F2 rev has worked. cut out and isolated 11 ug/ul Used in 3 PCRs, (pcr1)F1 mut1rc, (pcr2)Rev mut2rc and F1 Rev.
PCR worked. Isolated and pcr to get the final construct.
Metal Accumulation
- PCR to amplify fMT, SmtA with pre-RBS
- Redo PCR from yesterday as the PCR didnt proceed (hot start was manually)
- Run on gel and excise bands.
- Ligate pSB1AC3 with MymT, fMT and SmtA
- Do restriction of the vector and the inserts with EcoRI and SpeI.
- Do PCR clean-up kit to purify from the restriction enzymes.
- Check concentration and ligate o/n @ 4°C 100ng in a 3x overexcess of insert.
Vectors
Dry
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