BP Clonase Reaction and Electroporation Protocols
96 Well Format
PEG Precipitate PCR Fragment Figure
A. Aliquot 2 µl of PCR product (from a primary reaction) into 0.2 ml strip tubes.
B. Mix the following in a tube:
BP Clonase Cocktail / 1 X / 105X5 X BP Reaction Buffer / 2.0 µl / 210 µl
PCR product / 2.0 µl
pMK2010 (150 ng/µl in sdH2O) / 1.0 µl / 105 µl
Sterile deionized H20 / 4.0 µl / 420 µl
BP Clonase Enzyme Mix / 1.0 µl / 105 µl
C. Aliquot 8 µl of BP Clonase Cocktail into each tube containing 2 µl of PCR product (from a primary reaction). Remember to have a control tube that contains no PCR product.
D. Mix well and incubate at room temperature for 2-4 hours.
E. Add 3 µl Proteinase K (~7 mg/ml) or 1 µl Proteinase K (~20 mg/ml) to each tube, mix well and incubate at 37°C for 30 minutes.
F. Transfer 2 µl of BP Clonase treated PCR product into 50 µl of electroporation competent DH5α E. coli cells, place in a pre-chilled sterile electroporation cuvette with 0.1 cm gap and place in BTX TransPorator Plus (Harvard Apparatus Inc., Holliston, MA). Shock at a voltage of 1.5 kV. (link Preparation of electrocompentent cells)
G. Add 950 µl of SOC to the cuvette and move the cells from the cuvette into a 1.5 ml sterile Eppendorf tube, incubate at 37°C for 60-90 minutes.
H. Remove a 100 µl aliquot of each sample and spread on LBKan75 agar plate in order to obtain individual colonies. Incubate plates at 37°C overnight.
I. Electroporation cuvettes were cleaned using a cuvette washer, soaked in 70% ETOH for 5 min, allowed to dry, UV irradiated using a Fotodyne UV trans-illuminator for 5 min and stored at -20ºC. Electroporation cuvettes were recycled 10 times.
J. The remainder of the BP clonase reaction was stored at -80ºC and was used to recover additional clones when needed.
Invitrogen Gateway Cloning Manual: Gateway technology manual