Skim- milk Media

Materials 

triangle flask, balance, weighing paper, medicine spoon, mess cylinder, aluminium foil, autoclave, clean bench, petri dish, tryptone, yeast extract, sodium chloride, agar, skim milk, 3’ D.W , squeeze bottle , stirrer, magnetic bar , Alcohol Lamp

Method

1. LB plate / 70ml (for skim milk plate)

Tryptone           1g

Yeast extract      0.5g

Sodium chloride    1g

Agar             1.5g

3’ D.W 

- > Autoclave

2. 3% Skim milk LB plate / 100ml

LB plate                70ml

10% skim milk          30ml

- > Mix LB plate and Skim milk after autoclaved.

- > Pour the mixture 30ml/plate.

- > Store at 4℃ after sealing.

Prepare

① 10% skim milk / 150ml

Skim milk 15g

3’ D.W

- > Heating.

- > Autoclaved.

- > Store at 4℃

Protease culture

Materials 

soil sample, 3’ D.W, 50ml conical tube, 3% Skim milk LB plate, pipette, tip, micro tube, spreader, alcohol lamp, incubator, vortex

Method

1. Dry 3% Skim milk plate at Incubator 40- 60min.

2. Prepare the soil sample.

3. Add 3’ D.W (1:1)

4. Vortexing

5. Place at Room Temperature.

6. Spread supernatant 100µℓ onto Skim milk plate.

7. 37℃ Overnight.

Chromosomal DNA extraction

Materials 

TES buffer : 50mM Tris- Hcl(pH 7.5) , 10mM NaCl , 10mM EDTA , pH 7.5

TE  buffer : 10mM Tris (pH 8.0) , 0.1mM EDTA 

→ can be replaced to nuclease free water or Milli- Q water

Method

1. Grow cells in 2- 5ml broth to late log phase

2. Centrifugation the 1- 2ml cells Max RPM for 1min

3. Resuspend cells in 355ul TES buffer

4. Add : 40ul 10% SDS(Final concentration of 1%) and 5ul 10mg/ml Proteinase K (Final concentration  100ug/ml）

5．Incubate at 37°C for 30- 60 minutes of until the solution clears

6. Add 1:1 Phenol : Chloroform 

7. Centrifugation Max RPM , 5mins RT

8. Transfer the upper aqueous phase to a new tube.

9. Repeat 6~8

10. 

Ligation 

Materials 

micro tube, buffer, D.W, ligase, vector, insert

Method

1. vector : insert = 1 : 3 

insert(ng)= vector(ng) x insert(kb) x 3

vector(kb)

vector 100- 200ng

insertx

10x ligase buffer   1ul

ligase 1ul

D.W (up to 10ul)

2. incubate at RT for 2 hours. 

Transformation

Materials 

competent cell, sample DNA, ice, water bath, 5ml falcon tube, shaking incubator, pipette, tip, spreader, alcohol lamp, LB broth, LB plate, incubator

Method

1. competent cell 200㎕, incubate on ice for 30min, to thaw competent cell.

2. Add DNA sample 5ul into competent cell.

3. Incubate on ice for 30min

4. Heat shock in water bath at 42℃ for 1min and the put on ice for 2min.

5. Add LB broth 800㎕ and place in shacking incubate at 37℃ for 1h.

6. Spread 50㎕ onto LB plate

7. 37℃ overnight.

Prepare

① LB broth 37℃ Incubation

② LB plate 37℃ dry.

③ water bath 42℃ setting.

Cracking and Electrophoresis

Materials 

<Cracking>

cracking buffer, 10mM EDTA(pH 8.0), 4M KCl, micro tube, toothpick(autoclaved), vortex, heat block, ice, centrifuge, pipette, tip

<Electrophoresis>

0.8% agarose gel(+EtBr), 1X TAE, loading buffer, electrophoresis kit, UV illuminator

Method

<Cracking>

1. Add single colony in micro tube using toothpick.

2. Add 10mM EDTA 25㎕ and vortex.

3. Add cracking buffer 25㎕ and vortex.

4. Incubate at 70℃ for 5min.

5. Add 4M KCl 0.75㎕ and vortex.

6. Put on ice for 5min.

7. Centrifuge at 13000rpm, 4℃, 3min.

<Electrophoresis>

8. Load supernatant 10㎕ + loading buffer 2㎕ into 0.8% agarose gel.

9. After electrophoresis(100V, 20min), check under UV- illuminator

Prepare

① Cracking buffer / 50ml

5M NaOH2ml

10% SDS2.5ml

sucrose10g

3’DW 

② Heat block 70℃ setting

③ Centrifuge 4℃ setting