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 
- 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.
- 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 .
- 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 .
- 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
- 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
- Koch_Lab:Protocols/Oligonucleotide Annealing/Duplexes
- Silver: Oligonucleotide Inserts
- Annealing complementary primers
- Endy:Annealing complementary primers
- PNK Treatment of DNA Ends
- DNA ligation
July 28th 2009