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

Metal dependent promotors

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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 10 μL 10x Fast digest buffer (Fermentas) 2 μL SpeI (Fast digest, Fermentas) 2 μL EcoRI (Fast digest, Fermentas) 50 μL DNA (pSB3K3 or pSB1AC3) 36 μL MilliQ 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 Koch_Lab:Protocols/Oligonucleotide Annealing/Duplexes Silver: Oligonucleotide Inserts Annealing complementary primers Endy:Annealing complementary primers PNK Treatment of DNA Ends DNA ligation Results July 28th 2009