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

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*25 μL DNA (pSB3K3 or pSB1AC3)
*25 μL DNA (pSB3K3 or pSB1AC3)
*18 μL MilliQ
*18 μL MilliQ
Incubate 1 h @ 37 °C
Incubate 0.5 h @ 37 °C
<BR>
<BR>
Bring vectors to 2% agarose gel and cut out with scalpel, purify using gel purification kit to an end volume of 30 μL.
Bring vectors to 1% agarose gel and cut out with scalpel, purify using gel purification kit to an end volume of 30 μL.
(alternatively, [[Phosphatase treatment of linearized vector]])
(alternatively, [[Phosphatase treatment of linearized vector]])


Line 58: Line 58:
*Incubate @ 37 °C for 1.5 hours.  
*Incubate @ 37 °C for 1.5 hours.  
*Add 4 μL 0.5 M NaCl.  
*Add 4 μL 0.5 M NaCl.  
*Place in boiling water bath for 2 min., then remove water bath from the heat source and allow the reaction (still in the water bath) to cool to room temperature.
*Place in thermocycler (99 °C) for 3 min., and allow the reaction to cool to room temperature.


===Ligation===
===Ligation===

Revision as of 06:48, 28 July 2009

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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

  • 5 μL 10x Fast digest buffer (Fermentas)
  • 1 μL SpeI (Fast digest, Fermentas)
  • 1 μL EcoRI (Fast digest, Fermentas)
  • 25 μL DNA (pSB3K3 or pSB1AC3)
  • 18 μL MilliQ

Incubate 0.5 h @ 37 °C
Bring vectors to 1% agarose gel and cut out with scalpel, purify using gel purification kit to an end volume of 30 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

  • Add the vector (3 μL) into the annealing mix and then add ligase buffer (4 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 10 μL of ligation mixture to 50 μL of competent TOP10 cells
  • Heatshock, 45 sec. 42 °C
    • + control: 1 μL Uncut plasmid (pSB3K3 or pSB1AC3), - 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)
  • Grow ON @ 37 °C

Day 2

  • 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

Day 3

  • Isolate plasmids and perform restriction analysis

Protocols

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



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