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Team:Groningen/Notebook/15 September 2009
From 2009.igem.org
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The overnight cultures with LB-amp<sub>100</sub> medium of colonies E.coli TOP10 with | The overnight cultures with LB-amp<sub>100</sub> medium of colonies E.coli TOP10 with | ||
| + | |||
| + | |||
| + | [[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|150px| www.sigmaaldrich.com]] | ||
| + | |||
| + | '''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''' | ||
| + | |||
| + | {|cellpadding="2" cellspacing="1" border="4" | ||
| + | |'''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. | ||
| + | |||
| + | {|cellpadding="2" cellspacing="1" border="4" | ||
| + | |'''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 | ||
| + | |} | ||
| + | |||
| + | |||
| + | [[Image:15 no.1.jpg|340px]] [[Image:Generulers_1kb_marker_Fermentas.jpg]] | ||
| + | |||
| + | :→ From left to right: 1kB ladder, HmtA no.1, HmtA no.2, HmtA no.3, Empty Slot, pArsR-GVP, Empty Slot, pSB2K3 | ||
| + | |||
| + | |||
| + | '''Purification''' | ||
| + | |||
| + | [[Image:Zymoclean Gel DNA Recovery Kit (D4001) 2.jpg|thumb|150px| www.zymoresearch.com]] | ||
| + | |||
| + | |||
| + | |||
| + | :→ In step 7 the fragments were eluted in 12μL MQ and was stored on ice until use. | ||
| + | |||
| + | |||
| + | '''Concentration''' | ||
| + | |||
| + | |||
| + | '''Ligation''' | ||
| + | |||
| + | A total amount of vector of 100ng was used in a 1:3 ratio with insert. | ||
| + | |||
| + | :* 3 uL Ligase buffer | ||
| + | :* 1 uL 4 Ligase | ||
| + | :* 4 ul plasmid pSB1A2 pLacI digested with SpeI and PstI | ||
| + | :* uL GVP restricted with XbaI and PstI | ||
===Transporters=== | ===Transporters=== | ||
Revision as of 11:36, 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
Ligation
A total amount of vector of 100ng was used in a 1:3 ratio with insert.
- 3 uL Ligase buffer
- 1 uL 4 Ligase
- 4 ul plasmid pSB1A2 pLacI digested with SpeI and PstI
- uL GVP restricted with XbaI and PstI
Transporters
Metal Accumulation
Vectors
Dry
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