PEG Precipitation Protocol

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 105X 5 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