IGEM:Peking/2007/Switch: transformation

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Contents

Transformation

1. Put the competent cells into ice box as soon as their solution melts.

2. Add ligation system 10uL or plasmid 0.5uL into solution.

3. Keep in ice for 30min.

4. Heat shock in 42℃, JM109 and DH5alpha for 30s, BL21 for 120s, all other for 90s.

5. Put the competent cells into an ice box immediately for 2min.

6. Add 800uL LB media in a super clean bench.

7. Recover 160rpm for 30-45min.

8. If it is positive plasmid, smear 100uL solution directly onto plate. If it is ligation system, centrifuge for 5000rpm for 4min, discard the supernatant and smear the remainming onto the plate with antibiotic medium.

Preparation of Competent Cells

Steriliztion

Bottles, tubes and tips

SOB Preparation

950mL ddH2O

Tryptone 20g

Yeast Extract 5g

NaCl 0.5g

Add water to form a homogeneous solution

Sterilization

115℃ 20min

1L SOB + 10mL 2Mg2+ (1M MgCl2 + 1M MgSO4)

First time activation

Pick 1-10 single conlony from the plate

Add SOB,37℃ cultivated until saturation

TB preparation

PIPES 3.0g 10mM

CaCl2.2H2O 2.2g 15mM

KCl 18.6g 250mM

ddH2O 950ml

5N KOH -> pH 6.7

MnCl2.4H2O 10.9g 55mM

+ddH2O filtering the bacteria

Second time activation

1/100~1/30 inoculation

18℃ cultivated overnight

OD 0.4~0.8 (0.6 optimal)

centrifuge, tips, TB, tubes, pre-frozen tubes

ice for 10min

4℃ centrifuge,3000rpm,10min

1/3 v/v TB resuspend,ice for 10min

centrifuge for 1000rpm 10min

discard the supernatant, add 1/12.5 v/v TB resuspend

Add 7%v DMSO, ice for 10min

100uL/tube, quickly frozed using liquid nitrogen under -70℃

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