Team:KIT-Kyoto/Notebook-Protocol

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Revision as of 04:43, 21 September 2012






LB medium



*per kilogram
Bacto-tryptone10g
Bacto-yeast extract5g
NaCl10g
dH2O1000mL

1. Mix the reagents according to the previous components.
2. Adjust the pH (pH7.0) with NaOH.
3. Autoclave at 120℃ for 20min

LB plate



*per liter
Bacto-tryptone10g
Bacto-yeast extract5g
NaCl10g
dH2O1000mL

1. Mix the reagent according to the previous components.
2. Add NaOH and carry the enzymes pH7.0.
3. Add 15g Bacto agar and dissolve.
4. Autoclave at 120℃ 20min.
5. After cooling down approx. 60℃, add the suitable amounts of appropriate antibiotics.
6. Pour 20~30mL into laboratory dishes.

*Solution Ⅰ
 Glucose  50mM 
 EDTA  10mM 
 Tris-HCl  25mM 


*Solution Ⅱ
 NaOH  0.2N 
 SDS  1% 


*Solution Ⅲ
 K-acetate  5M 

Methods

Transformation into plasmid of E.coli.

1. Dissolve competent cell on ice.
2. Pour 100uL competent cell into 1.5mL tube, and mix with 1~5uL DNA.
3. Incubate for 30 minutes on ice.
4. Treat with heat shock the cells at 42℃ for 45 seconds.
5. Cool down on ice for 2 minutes.
6. Add 250uL S.O.C medium at room temperature.
7. Incubate at 37℃ with shaking for 1 hour.
8. Spread a suitable amount of the cells on LB plate (containing appropriate antibiotics).
9. Incubate overnight at 37℃.

Picking up a single colony

Pick up the colony grown on the LB plate by a platinum stick and put in 2.5mL of LB medium (containing appropriate antibiotics), and cultivate cells at 37˚C.

Miniprep by Alkaline-SDS method.



1. Spin 1.5 mL of culture in microcentrifuge tube at 12,000 rpm for 5 min. at 4 ˚C. Discard the supernatant
 and spin again for 2 min. Remove remaining liquid carefully.
2. Resuspend the bacterial cell pellet in 100 μL of solution I previously cooled on ice.
3. After 4 min. at room temperature, add 200 μL of solution II and mix by inverting several times.
4. Keep on ice for 4 min.
6. Add 160 μL of solution III and mix gently by inverting several times. Keep on ice for 5 min.
7. Centrifuge at 12,000 rpm for 10 min. at 4˚C
8. Transfer supernatant to new tube.
9. Add the same volume of phenol/chloroform and vortex well. Centrifuge at 12,000 rpm for 2 min at room
 temperature.
10. Transfer top clear layer to new tube. Add the same volume of diethyl ether and vortex. Centrifuge at
 12,000 rpm for 2 min. at room temperature.
11. Transfer the top clear layer to new tube. Add 2X volume of ethanol and vortex. Centrifuge at 12,000 rpm
 for 10 min. at room temperature.
12. Remove supernatant and add 1 mL of 70% ethanol and mix gently. Centrifuge at 12,000 rpm for 5 min at
 room temperature.
13. Remove supernatant and dry in vacuo. Dissolve pellet in 20 μL TE.

Miniprep by QIA prep Spin Miniprep Kit



1. Spin 1.5 mL of culture in microcentrifuge tube at 10,000 rpm for 5 min. at 4˚C. Discard the supernatant
 and spin again for 2 min. Remove remaining liquid carefully.
2. Resuspend the bacterial cell pellet in 250 μL of buffr P1.
3. Keep at room temperature for 4 min.
4. Add 250 μL of buffer P2 and mix by inverting quickly.
5. Keep on ice for 4 min.
6. Centrifuge at 13,000 rpm for 10 min. at 4˚C.
7. Apply supernatant to QIAprepspin column.
8. Centrifuge at 10,000 rpm for 40 sec. at room temperature. Discard the flow-through.
9. Add 500 μL of buffer PB. Centrifuge at 10,000 rpm for 40 sec. at room temperature. Discard the flow-
 through.
10. Add 750 μL of buffer PE. Centrifuge at 10,000 rpm for 40 sec. at room temperature. Discard the flow-
 through.
11. Centrifuge again at 10,000 rpm for 1 min. at room temperature. Set a column on new 1.5 mL tube.
12. Add 30 μL of buffer EB and keep at room temperature for 1 min. Centrifuge at 10,000 rpm for 1 min at
 room temperature.
13. Recover purified DNA in 1.5 mL tube.

Midiprep by Pure LinkTM HiPure Midiprep Kit



1. Centrifuge (4000xg,4°C,10min) E. coli culture in LB medium (50 mL) and remove the supernatant
2. Mix with suspension buffer (4mL) including RNase (20 micro g/mL) and the pellet
3. Add Lysis buffer (4mL) and turn upside and down several times in order to mix well
4. Incubate at room temperature for 5min
5. Add precipitation buffer (4mL) and shake the tube quickly
6. Centrifuge and remove the supernatant (15000 x g, room temperature, 10 min)
7. Transfer the supernatant to balanced column and elute out the solution by gravity
8. Wash the column twice with Wash buffer (10 mL) and elute out the solution as gravitation every after
 washing.
9. Put sterilized 15 mL centrifuge tube under the column
10. Add Elution buffer (5 mL) to the column and elute out the solution by gravity
11. Add isopropanol (3.5 mL) to the centrifuge tube and mix well
12. Centrifuge and remove the supernatant (15000 x g, 4°C, 30min)
13. Add 70% ethanol and mix well
14. Centrifuge and remove the supernatant (15000 x g, 4°C, 5min)
15. Dehydrated ,then suspend purified DNA into TE buffer (200 uL)

Purification of DNA from gel by QIA quick Gel Extraction Kit



1. Cut out the DNA fragment from agarose gel and put it in a 1.5mL tube and weigh the gel
2 .Add Buffer QG (x 3 volume of the gel weight)
3 .Vortex every 2 - 3 minutes to dissolve the gel completely
4 . Add isopropanol (equal weight to the gel, 100uL per 100mg)
5 . Trabsfer to spin column
6 . Centrifuge (10000rpm, room temperature,1min)
7 . Remove the solution
8 . Add Buffer PE (750 uL) and centrifuge (10000rpm,room temperature,1min)
9 . Remove the solution and centrifuge (17900 x g, room temperature, 1min)
10. Set a new 1.5 tube under the column, add TE (30uL) and centrifuge (10000 rpm, room temperature,1min)
11. Collect the purified DNA

BP reaction by Invitrogen gateway system



1. PCR using primers containing the attB sequence.
2. Purify PCR product.
3. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:

attB PCR product75 ng/reaction (1-7 μL)
pDONR vector150ng/reaction (1 μL)
TE Bufferto 8 μL

4. Vortex BP ClonaseII enzyme mix briefly. Add 1 - 2 μL to the components above and mix well by vortexing
 and spin them down.
5. Incubate reaction at 25˚C for more than 1 hour.
6. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly.
7. Incubate at 37˚C for 10 minutes.

LR reaction by Invitrogen gateway system



1. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:

Entry clones50-150 ng/reaction (1-7 μL)
Destination vector150ng/reaction (1 μL)
TE Bufferto 8 − 9 μL

2. Vortex LR ClonaseII enzyme mix briefly. Add 1 – 2 μL, to the components above
 and mix well by vortexing and spin down.
3. Incubate reaction at 25˚C for 16 hours.
4. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly.
5. Incubate at 37˚C for 10 minutes.