Team:Groningen/Protocols

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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

(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: pSB3K3 & pSB1AC3

Incubate 0.5 h @ 37 °C
Bring vectors to 1% agarose gel and cut out with scalpel, purify using gel purification kit (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 EcoRI (Non-fast digest)
    • 1 μL 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 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:


  • (ANTI-SENSE) Mix:
  • 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 pSB1AC3 or pSB3K3 carrying ccdB deathgene
  • 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 (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
  1. 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.
  2. Cells are divided in 50 μL aliquotes
  3. 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

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

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

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