Team:Groningen/Protocols
From 2009.igem.org
m |
m |
||
Line 1: | Line 1: | ||
{{Team:Groningen/Header}} | {{Team:Groningen/Header}} | ||
[[Category:Team:Groningen]] | [[Category:Team:Groningen]] | ||
+ | [[Category:Protocol]] | ||
=Protocols= | =Protocols= | ||
==Cloning== | ==Cloning== | ||
Line 43: | Line 44: | ||
<sup>*</sup>End volume determines concentration, variations are possible | <sup>*</sup>End volume determines concentration, variations are possible | ||
===Annealing synthetic oligo’s=== | ===Annealing synthetic oligo’s=== | ||
+ | ====Protocol #2==== | ||
+ | <u>Phosphorylation</u> | ||
+ | *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 | ||
+ | |||
+ | <u.>Annealing</u> | ||
+ | *Mix | ||
+ | ** 8 μL FW Phosphorylation mix | ||
+ | ** 8 μL REV Phosphorylation mix | ||
+ | ** 4 μL 10 mM NaCl {{todo|low?}} | ||
+ | ** 20 μL MilliQ | ||
+ | *Procedure | ||
+ | ** Put reactions in (close to) boiling water | ||
+ | ** Let it cool down naturally to ~35 °C | ||
+ | |||
+ | <u> Restriction vector</u> | ||
+ | *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 | ||
+ | |||
+ | <u>Ligation of Metal sensitive promoters in vector</u> | ||
+ | *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==== | ====Protocol #3==== | ||
<u>Restriction vectors</u> | <u>Restriction vectors</u> | ||
Line 110: | Line 156: | ||
*If enough transformants, inoculate 3 - 5 colonies in an ON culture | *If enough transformants, inoculate 3 - 5 colonies in an ON culture | ||
**Alternatively perform colony PCR | **Alternatively perform colony PCR | ||
+ | |||
+ | |||
===Ligation=== | ===Ligation=== | ||
* Ligation | * Ligation | ||
Line 135: | Line 183: | ||
===Restriction analysis=== | ===Restriction analysis=== | ||
=List of solutions= | =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 | ||
==Antibiotics== | ==Antibiotics== | ||
===[http://openwetware.org/wiki/Ampicillin Ampicillin]=== | ===[http://openwetware.org/wiki/Ampicillin Ampicillin]=== | ||
Line 152: | Line 209: | ||
* Filter sterilize 0.2 μm filter and aliquot | * Filter sterilize 0.2 μm filter and aliquot | ||
* Store -20 °C | * Store -20 °C | ||
+ | ==Chemicals== | ||
+ | ===0.1 M CaCl<sub>2</sub>=== | ||
+ | *0.3319 g CaCl<sub>2</sub> | ||
+ | *Dissolve in 30 mL demi water | ||
{{Team:Groningen/Footer}} | {{Team:Groningen/Footer}} |
Revision as of 12:14, 17 October 2009
Protocols
|
---|
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 #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
- 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
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
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
Chemicals
0.1 M CaCl2
- 0.3319 g CaCl2
- Dissolve in 30 mL demi water