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Team:Groningen/Notebook/15 September 2009
From 2009.igem.org
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'''Concentration''' | '''Concentration''' | ||
| + | |||
| + | '''Concentrations''' | ||
| + | |||
| + | {|cellpadding="2" cellspacing="1" border="4" | ||
| + | |'''Plasmid''' | ||
| + | |'''Conc. ng/μL''' | ||
| + | |'''260/280 | ||
| + | |'''260/230 ''' | ||
| + | |'''-20 box (michael | ||
| + | |'''Restriction Control''' | ||
| + | |- | ||
| + | |pArsR-GVP (E/P) | ||
| + | |36.1 | ||
| + | |1.91 | ||
| + | |0.17 | ||
| + | |x | ||
| + | |Yes (EcoRI/PstI) | ||
| + | |- | ||
| + | |pSB2K3 (E/P) | ||
| + | |58.0 | ||
| + | |1.93 | ||
| + | |0.13 | ||
| + | |x | ||
| + | |Yes (EcoRI/PstI) | ||
| + | |} | ||
Revision as of 11:42, 15 September 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
Planning
- → TODO work out the wiki page for GVP
- → TODO make a doodle for presentation planning (1-19 oct.)
- → TODO media attention
- → TODO
Plates
The
| Name | Plasmid Used | Antibiotics on Plasmid | No. of Colonies | Date |
| GlpF no.1 (low) | pSB1AC3 | Ampicillin | 0 | 14 sept. |
| GlpF no.1 (high) | pSB1AC3 | Ampicillin | 2 | 14 sept. |
| GlpF no.2 (low) | pSB1AC3 | Ampicillin | 1 | 14 sept. |
| GlpF no.2 (high) | pSB1AC3 | Ampicillin | 1 | 14 sept. |
| pLacI (low) | pSB1AC3 | Ampicillin | ~30 | 14 sept. |
| pLacI (high) | pSB1AC3 | Ampicillin | ~80 | 14 sept. |
| pLacI-GVP no.1 (low) | pSB1A2 | Ampicillin | 0 | 14 sept. |
| pLacI-GVP no.1 (high) | pSB1A2 | Ampicillin | 4 | 14 sept. |
| pLacI-GVP no.2 (low) | pSB1A2 | Ampicillin | 0 | 14 sept. |
| pLacI-GVP no.2 (high) | pSB1A2 | Ampicillin | 9 | 14 sept. |
| Positive (J23101) (high) | J61002 | Ampicillin | ~1500 | 14 sept. |
| Negative (MQ) (high) | MQ | Ampicillin | 0 | 14 sept. |
- → The plates showed a low amount of colonies, and also red in the case of the positive plate.
- → The two plates with stripes of pZntR-GVP (pSB2K3) and pCueO-GVP (pSB2K3) culture for glycerol stocks showed growth and some single colonies.
- → All plates were stored in the fridge for further use.
Cultures
The overnight cultures with LB-amp100 medium of colonies E.coli TOP10 with
Plasmid isolation
Plasmid isolation was performed on the cultures of E.coli TOP10 containing the above mentioned plasmids with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit".
- From each tube 5 to 10mL of culture was collected in a 2.0mL cup (tubes from pArsR-GVP, GVP and pNL29 were combined), and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
- Plasmids were eluted with 30μL MQ and stored in the fridge
Concentrations
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| pArsR-GVP (J61035) | 108.9 | 1.83 | 1.91 | x | Yes (EcoRI/PstI) |
| GVP (J61035) | 469.9 | 1.83 | 2.35 | x | x |
| pNL29 | 18.1 | 1.83 | 1.83 | x | x |
| HmtA no.1 | 52.1 | 1.85 | 1.87 | x | Yes (PstI) |
| HmtA no.2 | 49.4 | 1.83 | 1.61 | x | Yes (PstI) |
| HmtA no.3 | 42.6 | 1.91 | 2.06 | x | Yes (PstI) |
Restriction for Assembly, and Control (HmtA)
The plasmids from the o.n. precultures of pArsR-GVP and pSB2K3 (earlier last week) were cut with PstI and EcoRI to cut out the entire part between the pre- and suffix. The plasmids with PstI were cut with PstI for control.
| Plasmid | Amount μL | MQ μL | Fast digest buffer | EcoRI fast digest enzyme | XbaI fast digest enzyme | SpeI fast digest enzyme | PstI fast digest enzyme |
| pArsR-GVP | 13.0 | 3.0 | 3.0 | 1.0 | x | x | 1.0 |
| pSB2K3 | 4.0 | 11.0 | 3.0 | 1.0 | x | x | 1.0 |
| HmtA no.1 | 16.0 | x | 3.0 | x | x | x | 1.0 |
| HmtA no.2 | 16.0 | x | 3.0 | x | x | x | 1.0 |
| HmtA no.3 | 16.0 | x | 3.0 | x | x | x | 1.0 |
- → From left to right: 1kB ladder, HmtA no.1, HmtA no.2, HmtA no.3, Empty Slot, pArsR-GVP, Empty Slot, pSB2K3
Purification
_2.jpg)
- → In step 7 the fragments were eluted in 12μL MQ and was stored on ice until use.
Concentration
Concentrations
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| pArsR-GVP (E/P) | 36.1 | 1.91 | 0.17 | x | Yes (EcoRI/PstI) |
| pSB2K3 (E/P) | 58.0 | 1.93 | 0.13 | x | Yes (EcoRI/PstI) |
Ligation
A total amount of vector of 100ng was used in a 1:3 ratio with insert.
- 3 uL Ligase buffer
- 1 uL T4 Ligase
- 9 uL MQ
- 4 uL plasmid pSB2K3 EcoRI/PstI
- 3 uL GVP restricted with EcoRI/PstI
Incubate:
- 25°C 50min.
- kept on ice for 10min.
Tranformation
- add 10uL of the 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
- → Positive control was BBa_J61002-J23101 plasmid, and negative control was MQ.
Transporters
Metal Accumulation
Vectors
Dry
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