Team:Groningen/Protocols
From 2009.igem.org
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m (→Restriction analysis) |
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===Colony PCR=== | ===Colony PCR=== | ||
===Restriction analysis=== | ===Restriction analysis=== | ||
+ | ==Measurements== | ||
+ | ===Metal uptake assay for <i>E. coli</i> (according to Kostal et al. 2004)=== | ||
+ | *Grow o/n culture of E. coli WT @ 30dg | ||
+ | **Use E. coli + control vector, E. coli + pArsR-RFP, E. coli + pLac-fMT | ||
+ | *Inoculate day culture 1:50, grow in 1L TB-Amp (100ml per time/[As(III)] sample) | ||
+ | **Take OD600 samples every 1-1.5 hrs of E. coli + pLac-fMT. | ||
+ | **Induce E. coli + pLac-fMT at OD600 ~0.6 with 0.5mM IPTG. | ||
+ | *Harvest the cells in stationary phase (after ~30hr) by spinning down @ 4000rpm for 20min in Sorval centrifuge. | ||
+ | *Wash 2 times with TB74S buffer. | ||
+ | *Resuspend in prewarmed (30 C) TB74S buffer upto a OD of ~25 | ||
+ | **Take a 1ml sample in small alluminium boxes and dry @ 104dg for >4 hrs. | ||
+ | **Afterwards measure the dry weight of the sample and calculate the weight/volume of the entire sample. | ||
+ | *For the concentration range: | ||
+ | **Incubate 5 samples (of same time point) for 1hr @ 30dg with 0μM, 10 μM, 20 μM, 50 μM and 100 μM As(III). | ||
+ | *For the concentration range: | ||
+ | **Incubate 5 samples (of same concentration) @ 30dg with 10 or 100μM As(III) for 0, 10, 20, 40, 60 min. | ||
+ | *Harvest cells by spinning down. | ||
+ | *Wash the cells with TB74S buffer. | ||
+ | *Resuspend in 10ml ddH2O. | ||
+ | *Dry sample @ 65dg for 2days. | ||
+ | *Store at fridge or -80dg freezer. | ||
+ | *Determine the amount of As(III) in the cell at different stages and at different uptake concentrations using ICP-MS. | ||
+ | |||
+ | ====Analysis of arsenic concentration of ICP-MS==== | ||
+ | |||
+ | *Weigh 0.1g dried <i>E. coli</i> cells. | ||
+ | *Add 5 ml 65% nitric acid. | ||
+ | *For destruction the following microwave program was used: | ||
+ | |||
+ | {|cellpadding="2" cellspacing="1" border="4" | ||
+ | |''' ''' | ||
+ | |'''Stage 1''' | ||
+ | |'''Stage 2''' | ||
+ | |- | ||
+ | |Power(max) | ||
+ | |1200 | ||
+ | |1200 | ||
+ | |- | ||
+ | |Power(%) | ||
+ | |100 | ||
+ | |100 | ||
+ | |- | ||
+ | |Ramp(min) | ||
+ | |15 | ||
+ | |15 | ||
+ | |- | ||
+ | |Hold(min) | ||
+ | |0 | ||
+ | |30 | ||
+ | |- | ||
+ | |Temp(graden C) | ||
+ | |140 | ||
+ | |210 | ||
+ | |} | ||
+ | |||
+ | |||
+ | *Let the samples cool down. | ||
+ | *Dilute the samples by adding ddH2O upto 50 ml. | ||
+ | *If needed, spin down 15min @ 4000rpm in a Sorvall centrifuge. | ||
+ | *Measure the arsenic concentration by ICP-MS using both the standard mode (shows interference peak from multi-atomic molecule argon-chloride with the arsenic peak) and the collusion cell technology mode (doesn’t show the interference peak but has a 10x lower resolution than standard mode). | ||
+ | **Use a standard curve between 0-10µg As/L and 0-100µg As/L using a certified 1000ppm (mg/L) stock | ||
+ | |||
=List of solutions= | =List of solutions= | ||
==Media== | ==Media== |
Revision as of 13:14, 17 October 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Protocols
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Protocols
Cloning
[http://openwetware.org/wiki/PCR PCR]
21 μL mastermix*
1 μL forward primer
1 μL reverse primer
1 μL template
1 μL Taq polymerase
*master mix contains:
100 μL Taq NH4
8 μL dNTP's
80 μL MgCl
652 μL MilliQ water
PCR Reaction
- Hotstart
- 95 °C, 2 min.
- 25 cycles
- 95 °C, 30 sec.
- 61 °C, 20 sec.
- 72 °C, 1.5 min.
- End
- 72 °C, 10 min.
- 4 °C, ∞
Plasmid isolation
(NucleoSpin® Plasmid, Machery nagel])
(GeneElute™ Plasmid Miniprep Kit, Sigma-Aldrich)
Restriction
- Cut plasmids containing promotor with SpeI and PstI
- 1 μL 10x fast digest buffer
- 0.5 μL SpeI
- 0.5 μL PstI
- 8 μL Vector
- Incubate 1 h @ 37 °C
- Purify cut plasmid using PCR clean up kit
Restriction vectors
Vectors: [http://partsregistry.org/Part:pSB3K3 pSB3K3] & [http://partsregistry.org/Part:pSB1AC3 pSB1AC3]
- 10 μL 10x Fast digest buffer ([http://www.fermentas.com/ Fermentas])
- 2 μL [http://www.fermentas.com/catalog/re/fastbcui.htm SpeI] (Fast digest, [http://www.fermentas.com/ Fermentas])
- 2 μL [http://www.fermentas.com/catalog/re/fastecori.htm EcoRI] (Fast digest, [http://www.fermentas.com/ Fermentas])
- 50 μL DNA ([http://partsregistry.org/Part:pSB3K3 pSB3K3] or [http://partsregistry.org/Part:pSB1AC3 pSB1AC3])
- 36 μL MilliQ
Incubate 0.5 h @ 37 °C
Bring vectors to 1% agarose gel and cut out with scalpel, purify using gel purification kit ([http://www.mn-net.com/Products/NucleicAcidPurification/DNAcleanup/NucleoSpinExtractII/tabid/1452/language/en-US/Default.aspx NucleoSpin® Extract II, Machery nagel]) to an end volume of 50 μL.
(alternatively, Phosphatase treatment of linearized vector)
Gel isolation
- Put restriction on 1% Agarose (TBE) gel
- Cut band of desired bp out
- Purify using gel purification kit
- End volume 30 μL*
*End volume determines concentration, variations are possible
Annealing synthetic oligo’s
Protocol #2
Phosphorylation
- Mix
- 1 μL PNK
- 1 μL T4 DNA Ligase buffer
- 8 μL Oligo’s (FW and REV seperate reactions)
100x diluted oligo’s (1 μM)
- Procedure
- 30 min 37 °C
- 20 min 65 °C
Annealing
- Mix
- 8 μL FW Phosphorylation mix
- 8 μL REV Phosphorylation mix
- 4 μL 10 mM NaCl low?
- 20 μL MilliQ
- Procedure
- Put reactions in (close to) boiling water
- Let it cool down naturally to ~35 °C
Restriction vector
- Mix
- 1 μL EcoRI
- 1 μL XbaI
- 2 μL Fast digest buffer (10x)
- 8 μL Vector DNA
- 8 μL MilliQ
- Procedure
- 37 °C, 30 min. incubation
- Heat inactivation EcoRI and XbaI
5 min. 80 °C 15 min. 65 °C
Ligation of Metal sensitive promoters in vector
- Mix
- 2 μL T4 Ligase Buffer
- 0.5 μL T4 Ligase
- 1 μL Restriction product (unpurified)
- 1 μL Annealed oligo’s
- 18 μL MilliQ
- Procedure
- Incubation 16 °C, 1 hour
- Heat inactivation Ligase
65 °C, 10 min.
Protocol #3
Restriction vectors
- Mix
- 2 μL Tango digest buffer (Fermentas)
- 16 μL vector
- 1 μL [http://www.fermentas.com/catalog/re/ecori.htm EcoRI] (Non-fast digest)
- 1 μL [http://www.fermentas.com/catalog/re/bcui.htm SpeI] (Non-fast digest)
- Incubate 1.5 h @ 37 °C
- Put to 1% agarose (1xTBE)
- Isolate ~3000 bp vector
- Check for presence of cut out [http://partsregistry.org/Part:BBa_P1010:Design ccdB death gene] (~600 bp)
- Purify vector using gel purification kit
- End volume 30 μL H2O (MilliQ)
- Determine concentration using nanodrop
- Store to 4 °C until ligation
Phosphorylation of 5' ends & hybridization [1]
- (SENSE) Mix:
- 3 μL 100 µM sense oligo
- 1 μL 10 x PNK (polynucleotide kinase) buffer ([http://www.fermentas.com/catalog/modifyingenzymes/t4polynucleotidekinase.htm Fermentas Buffer A])
- 2 μL 10mM ATP
- 1 μL [http://www.fermentas.com/catalog/modifyingenzymes/t4polynucleotidekinase.htm T4 polynucleotide kinase (PNK)]
- 3 μL MilliQ
- (for selfcloser control, do not add oligo's. Instead 6 μL MilliQ in total)
- (ANTI-SENSE) Mix:
- 3 μL 100 µM anti-sense oligo
- 1 μL 10 x PNK (polynucleotide kinase) buffer ([http://www.fermentas.com/catalog/modifyingenzymes/t4polynucleotidekinase.htm Fermentas Buffer A])
- 2 μL 10mM ATP
- 1 μL [http://www.fermentas.com/catalog/modifyingenzymes/t4polynucleotidekinase.htm T4 polynucleotide kinase (PNK)]
- 3 μL MilliQ
- (for selfcloser control, do not add oligo's. Instead 6 μL MilliQ in total)
- Incubate @ 37 °C for 1.5 hours.
- Mix
- 10 μL Sense mixture
- 10 μL Anti-sense mixture
- 3 μL 0.5 M NaCl
- Place in boiling water for 3 min., and allow the reaction to cool to room temperature.
Ligation
- Mix
- 1 μL T4 ligase buffer
- 7.5 μL restricted vector (purified from gel)
- 1 μL annealing mix
- 0.5 μL T4 ligase
- Incubate
- 1h RT
- or
- ON @ 4 °C
Transformation
- Add 5 μL of ligation mixture to 50 μL of TOP10 chemically competent cells
- Heatshock, 45 sec. 42 °C
- + control: 1 μL pSB3K3-high or pSB1AC3-high plasmid, - control: 1 μL MilliQ
- Alternatively a single cut plasmid can be taken as a ligation control
- Alternative - control: 1 μL [http://partsregistry.org/Part:pSB1AC3 pSB1AC3] or [http://partsregistry.org/Part:pSB3K3 pSB3K3] carrying ccdB deathgene
- Incubate 1 h @ 37 °C, 200 RPM
- Plate out on LB-agar + Kanamycin (30 μg/ml for [http://partsregistry.org/Part:pSB3K3 pSB3K3]) or Ampicillin (100 μg/mL for [http://partsregistry.org/Part:pSB1AC3 pSB1AC3])
- Plate out 50 μL & 200 μL of cell suspension
- Grow ON @ 37 °C
Checking transformations
- See if - control is empty for functioning antibiotics and death gene
- See how many colonies on + control for functioning competent cells
- See how many selfclosers and compare to samples (>10x on sample vs. selfcloser)
- If enough transformants, inoculate 3 - 5 colonies in an ON culture
- Alternatively perform colony PCR
Ligation
- Ligation
- μL ligation buffer
- 1 μL ligase
- μL vector
- μL insert
- Incubate
Making competent cells
Competent cells: TOP10 & DB3.1
- 10 mL ON culture is used to inoculate LB, 100 μL ON culture per 20 mL*
- Cultures are grown @ 37 °C until an OD600 of 0.2 ~ 0.3 is reached.
- Cultures are spinned down 5 min. @ 4000 rpm, 4 °C
- Supernatant is removed and pellet (per 20 mL culture) is resuspended in 5 mL chilled 0.1 M CaCl2
- Suspension is incubated on ice for 10 min.
- Suspensions are spinned down 5' @ 4000 rpm, 4 °C
- Supernatant is removed and pellet is resuspended in 1770 μL chilled 0.1 M CaCl2 and supplemented with 230 μL 87% glycerol prior to making aliquots.
- Cells are divided in 50 μL aliquotes
- Cells are snapfrozen in liquid nitrogen and stored @ -80 °C
* Cultures should be grown in the ratio 1:5 (medium:air), so 10 mL culture in a 50 mL greiner tube.
Transformation
Quality control
Colony PCR
Restriction analysis
Measurements
Metal uptake assay for E. coli (according to Kostal et al. 2004)
- Grow o/n culture of E. coli WT @ 30dg
- Use E. coli + control vector, E. coli + pArsR-RFP, E. coli + pLac-fMT
- Inoculate day culture 1:50, grow in 1L TB-Amp (100ml per time/[As(III)] sample)
- Take OD600 samples every 1-1.5 hrs of E. coli + pLac-fMT.
- Induce E. coli + pLac-fMT at OD600 ~0.6 with 0.5mM IPTG.
- Harvest the cells in stationary phase (after ~30hr) by spinning down @ 4000rpm for 20min in Sorval centrifuge.
- Wash 2 times with TB74S buffer.
- Resuspend in prewarmed (30 C) TB74S buffer upto a OD of ~25
- Take a 1ml sample in small alluminium boxes and dry @ 104dg for >4 hrs.
- Afterwards measure the dry weight of the sample and calculate the weight/volume of the entire sample.
- For the concentration range:
- Incubate 5 samples (of same time point) for 1hr @ 30dg with 0μM, 10 μM, 20 μM, 50 μM and 100 μM As(III).
- For the concentration range:
- Incubate 5 samples (of same concentration) @ 30dg with 10 or 100μM As(III) for 0, 10, 20, 40, 60 min.
- Harvest cells by spinning down.
- Wash the cells with TB74S buffer.
- Resuspend in 10ml ddH2O.
- Dry sample @ 65dg for 2days.
- Store at fridge or -80dg freezer.
- Determine the amount of As(III) in the cell at different stages and at different uptake concentrations using ICP-MS.
Analysis of arsenic concentration of ICP-MS
- Weigh 0.1g dried E. coli cells.
- Add 5 ml 65% nitric acid.
- For destruction the following microwave program was used:
Stage 1 | Stage 2 | |
Power(max) | 1200 | 1200 |
Power(%) | 100 | 100 |
Ramp(min) | 15 | 15 |
Hold(min) | 0 | 30 |
Temp(graden C) | 140 | 210 |
- Let the samples cool down.
- Dilute the samples by adding ddH2O upto 50 ml.
- If needed, spin down 15min @ 4000rpm in a Sorvall centrifuge.
- Measure the arsenic concentration by ICP-MS using both the standard mode (shows interference peak from multi-atomic molecule argon-chloride with the arsenic peak) and the collusion cell technology mode (doesn’t show the interference peak but has a 10x lower resolution than standard mode).
- Use a standard curve between 0-10µg As/L and 0-100µg As/L using a certified 1000ppm (mg/L) stock
List of solutions
Media
LB(Agar)
- 10 g (Bacto)Trypton
- 10 g NaCl
- 5 g Yeast extract
- Dissolve in 1 L demi water
- (1.5% Agar)
- Autoclave
- Store @ 60 °C
TB medium
- 12g Bacto-Tryptone
- 24g Bacto-Yeast Extract
- 4ml Glycerol [87%]
- dissolve in 900ml demi water
- Separetely prepare 100 mL Kpi
- 0.17M KH2PO4 (mw=136.09g/mol) (6.94g/300ml)
- 0.72M K2HPO4 (mw=174.18g/mol) (7.62g/300ml)
- dissolve in demi water
- Autoclave and mix
Antibiotics
[http://openwetware.org/wiki/Ampicillin Ampicillin]
100 mg/ml Ampicillin (1000x) Stock
- 1 g of Ampicillin sodium salt in 10 mL of demiwater (or 50% EtOH)
- Add NaOH or KOH to allow the Ampicillin to dissolve
- Filter sterilize 0.2 μm filter and aliquot
- Store -20 °C
[http://openwetware.org/wiki/Chloramphenicol Chloramphenicol]
35 mg/ml Chloramphenicol (1000x) Stock
- 0.35 g in 10 mL 100% EtOH
- Filter sterilize 0.2 μm filter and aliquot
- Store -20 °C
[http://openwetware.org/wiki/Kanamycin Kanamycin]
50 mg/ml Kanamycin(1000x) Stock
- g in 10 mL
- Filter sterilize 0.2 μm filter and aliquot
- Store -20 °C
Chemicals
0.1 M CaCl2
- 0.3319 g CaCl2
- Dissolve in 30 mL demi water
0.15 M NaCL (Saline solution, 0.9% NaCl)
- 9 g NaCl
- Dissolve in 1 L demi water
TBE buffer
4 M NaOH
1 M HCl
TB74S Buffer
- 0.605 g Tris (5mM)
- 8.76 g NaCl (150mM)
- Dissolve in 1 L Demi water
- Set pH with HCl to pH 7.4
Sodium Arsenite (III)
- 100mM Na-As solution
- filter sterilize