Team:Groningen/Protocols
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{{Team:Groningen/Header}} | {{Team:Groningen/Header}} | ||
[[Category:Team:Groningen]] | [[Category:Team:Groningen]] | ||
- | + | =Protocols= | |
- | + | ==Cloning== | |
- | + | ===[http://openwetware.org/wiki/PCR PCR]=== | |
- | < | + | 21 μL mastermix<sup>*</sup><BR> |
- | < | + | 1 μL forward primer<BR> |
- | < | + | 1 μL reverse primer<BR> |
- | < | + | 1 μL template<BR> |
- | < | + | 1 μL Taq polymerase<BR> |
- | < | + | |
- | < | + | <sup>*</sup>master mix contains:<BR> |
- | < | + | 100 μL Taq NH<sub>4</sub><BR> |
- | < | + | 8 μL dNTP's <BR> |
- | < | + | 80 μL MgCl<BR> |
- | < | + | 652 μL MilliQ water<BR> |
- | < | + | <BR> |
- | < | + | <u><b>PCR Reaction</b></u> |
+ | * 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=== | ||
+ | ===Restriction=== | ||
+ | * Cut plasmids containing promotor with <i>SpeI</i> and <i>PstI</i> | ||
+ | ** 1 μL 10x fast digest buffer | ||
+ | ** 0.5 μL <i>SpeI</i> | ||
+ | ** 0.5 μL <i>PstI</i> | ||
+ | ** 8 μL Vector | ||
+ | * Incubate 1 h @ 37 °C | ||
+ | * Purify cut plasmid using PCR clean up kit | ||
+ | ===Gel isolation=== | ||
+ | * Put restriction on 1% Agarose (TBE) gel | ||
+ | ** Cut band of desired bp out | ||
+ | ** Purify using gel purification kit | ||
+ | *** End volume 30 μL<sup>*</sup> | ||
+ | <sup>*</sup>End volume determines concentration, variations are possible | ||
+ | ===Annealing synthetic oligo’s=== | ||
+ | ====Protocol #3==== | ||
+ | <u>Restriction vectors</u> | ||
+ | * Mix | ||
+ | ** 2 μL Tango digest buffer (Fermentas) | ||
+ | ** 16 μL vector | ||
+ | ** 1 μL [http://www.fermentas.com/catalog/re/ecori.htm <i>EcoRI</i>] (Non-fast digest) | ||
+ | ** 1 μL [http://www.fermentas.com/catalog/re/bcui.htm <i>SpeI</i>] (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 H<sub>2</sub>O (MilliQ) | ||
+ | ** Determine concentration using nanodrop | ||
+ | ** Store to 4 °C until ligation | ||
+ | ---- | ||
+ | <u>Phosphorylation of 5' ends & hybridization<SUP><FONT SIZE="-1">[[Silver: Oligonucleotide Inserts| [1]]]</FONT></SUP></u> | ||
+ | *(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) | ||
+ | <BR> | ||
+ | *(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. | ||
+ | ---- | ||
+ | <u>Ligation</u> | ||
+ | *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 | ||
+ | ---- | ||
+ | <u>Transformation</u> | ||
+ | *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 | ||
+ | ---- | ||
+ | <u>Checking transformations</u> | ||
+ | *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<sup>*</sup> | ||
+ | *Cultures are grown @ 37 °C until an OD<sub>600</sub> of 0.2 ~ 0.3 is reached. | ||
+ | *Cultures are spinned down 5 min. @ 4000 rpm, 4 °C | ||
+ | *Supernatant is removed and pellet 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 | ||
+ | **Pellets were left on ice for undetermined time due to lack of liquid nitrogen | ||
+ | *Supernatant is removed and pellet is resuspended in 2000 μL chilled 0.1 M CaCl2+10% glycerol prior to making aliquots. | ||
+ | *Cells are divided in 50 μL aliquotes | ||
+ | *Cells are stored @ -80 °C | ||
+ | <sup>*</sup> 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=== | ||
+ | =List of solutions= | ||
+ | ==Antibiotics== | ||
+ | ===[http://openwetware.org/wiki/Ampicillin Ampicillin]=== | ||
100 mg/ml Ampicillin (1000x) Stock | 100 mg/ml Ampicillin (1000x) Stock | ||
* 1 g of Ampicillin sodium salt in 10 mL of demiwater (or 50% EtOH) | * 1 g of Ampicillin sodium salt in 10 mL of demiwater (or 50% EtOH) | ||
Line 22: | Line 142: | ||
* Filter sterilize 0.2 μm filter and aliquot | * Filter sterilize 0.2 μm filter and aliquot | ||
* Store -20 °C | * Store -20 °C | ||
- | + | ===[http://openwetware.org/wiki/Chloramphenicol Chloramphenicol]=== | |
35 mg/ml Chloramphenicol (1000x) Stock | 35 mg/ml Chloramphenicol (1000x) Stock | ||
* 0.35 g in 10 mL 100% EtOH | * 0.35 g in 10 mL 100% EtOH | ||
* Filter sterilize 0.2 μm filter and aliquot | * Filter sterilize 0.2 μm filter and aliquot | ||
* Store -20 °C | * Store -20 °C | ||
- | + | ===[http://openwetware.org/wiki/Kanamycin Kanamycin]=== | |
50 mg/ml Kanamycin(1000x) Stock | 50 mg/ml Kanamycin(1000x) Stock | ||
* {{todo| g in 10 mL }} | * {{todo| g in 10 mL }} |
Revision as of 11:57, 17 October 2009
Protocols
|
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Protocols
Cloning
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
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
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 #3
Restriction vectors
- Mix
- Incubate 1.5 h @ 37 °C
- Put to 1% agarose (1xTBE)
- Isolate ~3000 bp vector
- Check for presence of cut out 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 (Fermentas Buffer A)
- 2 μL 10mM ATP
- 1 μL 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 (Fermentas Buffer A)
- 2 μL 10mM ATP
- 1 μL 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
- Incubate 1 h @ 37 °C, 200 RPM
- Plate out on LB-agar + Kanamycin (30 μg/ml for pSB3K3) or Ampicillin (100 μg/mL for 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 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
- Pellets were left on ice for undetermined time due to lack of liquid nitrogen
- Supernatant is removed and pellet is resuspended in 2000 μL chilled 0.1 M CaCl2+10% glycerol prior to making aliquots.
- Cells are divided in 50 μL aliquotes
- Cells are 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
List of solutions
Antibiotics
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
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
Kanamycin
50 mg/ml Kanamycin(1000x) Stock
- g in 10 mL
- Filter sterilize 0.2 μm filter and aliquot
- Store -20 °C