User:Wilfred J. Poppinga/Notebook/Wilfreds Project/2009/07/28

From OpenWetWare

Jump to: navigation, search
Metal dependent promotors Main project page
Previous entry      Next entry

M&M

Oligo's

'
Copper sensitive promotors Zinc sensitive promotors Arsenic sensitive promotors
CueO+RBS FW ZntR+RBS FW ArsRpromo+RBS FW
CueO+RBS REV ZntR+RBS REV ArsRpromo+RBS REV
CueO-RBS FW ZntR-RBS FW ArsRpromo-RBS FW
CueO-RBS REV ZntR-RBS REV ArsRpromo-RBS REV

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)

Phosphorylation of 5' ends & hybridization [1]

  • Mix:
    • 3 μL 100 µM sense oligo
    • 3 μL 100 µM anti-sense oligo
    • 3 μL 10 x PNK (polynucleotide kinase) buffer (Fermentas Buffer A)
    • 2 μL 10mM ATP
    • 2 μL T4 polynucleotide kinase (PNK)
    • 17 μL MilliQ
      • (for selfcloser control, do not add oligo's. Instead 23 μL MilliQ in total)

to give 30 μL total volume

  • Incubate @ 37 °C for 1.5 hours.
  • Add 4 μL 0.5 M NaCl.
  • Place in thermocycler (99 °C) for 3 min., and allow the reaction to cool to room temperature.

Ligation

  • Mix the vector (3 μL) and the annealing mix 1 μL and then add ligase buffer (1 μL) and ligase (1 μL) (and 4 μL MilliQ) once the mix gets close to room temperature. this should reduce the likelihood of insert multimers forming [2].
    • As the paired oligos cool, they will also form multimers of your insert. To release them, you should heat the mixture of your vector and insert DNA to about 65 °C and let it cool prior to adding ligase [2].

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
  • 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
  • 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

Plasmid isolation and quality control

  • Isolate plasmids and perform restriction analysis

Protocols

  1. Koch_Lab:Protocols/Oligonucleotide Annealing/Duplexes
  2. Silver: Oligonucleotide Inserts
  3. Annealing complementary primers
  4. Endy:Annealing complementary primers
  5. PNK Treatment of DNA Ends
  6. DNA ligation

Results

July 28th 2009



Personal tools