Cryo-Competent S.pombe cell for transformation

Aim

To easy and high-efficiency transformation.

Materials

SD medium

10x LiOAc TE LiOAc 33g

2M Tris (pH7.5) 25ml

0.5M EDTA (pH8.0) 10ml

mess up to 500ml by DW

autoclave

1x LiOAc/TE with 30% Glycerool

10x LiOAc/TE 5ml

Glycerol 15ml

H2O up to 50ml

50% PEG in LiOAc/TE

10x LiOAc/TE 1ml

PEG#4000 5g

H2O up to 10ml

Methods

Competent cell preparation

Cell is cultured with 100ml SD media in the flask at 32℃(if cell is ts, 25℃ incubation should be better).

1.0x10^7cells/ml

The culture is placed on ice for 15min.

Cells are collected to a 50 ml tube by 3000rpm and wash twice with 1 ml ice-cold water.

Cell pellet is suspend into 1ml ice-cold 1xLiOAC/TE-Glycerol (1.0x 10^9 cels/ml).

50ul suspensions are dispensed into 1.5 ml tubes.

tubes are placed on ice for 30min.

Cells are frozen and stored in the -80℃ deep freezer.

Transformation

Frozen competent cells are quickly thawed in a 40℃ heat block for 30sec~2min.

add 5ul a carrier DNA (10mg/ml) and a plasmid or PCRed DNA.

add 145ul 50% PEG in 1xLiOAc/TE, vortex well.

incubate at 42℃ for 15min.

3000rpm for 1min and remove the sup.

pellet was re-suspend with media or water.

cell suspension is spread onto the plate.

Remarks

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