Team:NTHU Taiwan/Protocol
Protocol
Routine Bacterial Culture (Burkholderia acidipaludis)
A. Liquid culture
1.Prepare 5 bacterial culture tubes .
2.Move in hood .
3.Add 3 ml of PYM medium .
4.Add 10 ul of last liquid culture . ( record which one )
5.Incubate at 25 degree Celsius .
B. Solid culture
1.Prepare 2 PYM agar plate .
2.Add 5 ul of last liquid culture . ( should use the same one )
3.Use streaking method to isolate single colonies .
4.Incubate at 25 degree Celsius .
Bacterial Culture (E.coli)
A. Liquid culture
1. Prepare enough bacterial culture tubes .
2. Move in hood .
3. Add 2ml of LB medium .
4. Add 10 ul of E.coli contained medium .
5. Incubate at 37 degree Celsius, shake .
Preparation of LB medium
Material:
1 L of LB medium contained
10 g Tryptone
5 g Yeast extract
10 g NaCl
15 g Bacterial Culture Agar ( solid medium )
1 L double filtrate water
Method:
1. Prepare a serum bottle .
2. Add 12.5 g of LB powder .
3. Add 7.5 g of agar powder . ( if solid medium is needed )
4. Add 500 ml double filtrated water .
5. Mix it well .
6. Autoclave .
7. Wait till the solid medium cool down . ( only solid medium )
8. Pour into culture dish in hood . ( only solid medium )
9. Wait till the medium cool down to room temperature .
10. Store at 4 degree Celsius .
Preparation of PYM medium
Material:
1 L of PYM medium contained
10 g Polypeptone
2 g Yeast extract
1 g MgSO4
15 g Bacterial Culture Agar ( solid medium )
1 L double filtrate water
Method:
1. Prepare a serum bottle .
2. Add 5 g of Polypepton .
3. Add 1 g of Yeast extract .
4. Add 0.5 g of MgSO4
5. Add 7.5 g of agar powder . ( if solid medium is needed )
6. Add 500 ml double filtrated water .
7. Mix it well .
8. Autoclave
9. Wait till the solid medium cool down . ( only solid medium )
10. Pour into culture dish in hood . ( only solid medium )
11. Wait till the medium cool down to room temperature .
12. Store at 4 degree Celsius .
Preparation of LB medium with CAM
1. Prepare a serum bottle .
2. Add 12.5 g of LB powder .
3. Add 7.5 g of agar powder . ( if solid medium is needed )
4. Add 500 ml double filtrated water .
5. Mix it well .
6. Autoclave
7. Wait till the medium cool down .
8. Add 10 mg of CAM . ( Chloramphenicol )
( the concentration of CAM should be 20 mg / L )
9. Pour into culture dish in hood . ( only solid medium )
10. Wait till the medium cool down to room temperature .
11. Store at 4 degree Celsius .
Transformation (For E.coli DH5alpha strain)
1. Prepare eppendorfs .
2. Add 1 ul of target DNA and 10 ul of DH5alpha competent cells .
( compedent cells store at – 80 degree Celsius and can only use once )
For amplify experiment , add 1 ul DNA and 10 ul competent cells .
For ligation experiment , add 3 ul DNA and 30 ul competent cells .
For PCR product , add 5 ul DNA and 50 competent cells .
3. Place it on ice for 20 minutes .
4. Heatshock it at 42 degree Celsius for 45 seconds .
5. Place it on ice for 2 minutes .
6. Add 200 ul LB medium .
7. Incubate at 37 degree Celsius for 1 hour .
8. At the same time , preheating a LB plate with CAM in 37 degree Celsius incubator.
9. Move in hood .
10. Put 5-10 small sterile beads on the plate .
11. Pour bacteria on beads and shake plate .
12. Discard beads .
13. Incubate at 37 degree Celsius overnight .
( or 25 degree Celsius for 48 hours )
Transformation (For E.coli MG1655 strain)
1. Prepare eppendorfs .
2. Add 1 ul of target DNA and 50 ul of DH5alpha compedent cells .
( compedent cells store at – 80 degree Celsius and can only use once )
3. Place it on ice for 20 minutes .
4. Heatshock it at 42 degree Celsius for 60 seconds .
5. Place it on ice for 5 minutes .
6. Add 150 ul LB medium .
7. Incubate at 37 degree Celsius for 1 hour .
8. At the same time , preheating a LB plate with CAM in 37 degree Celsius incubator.
9. Move in hood .
10. Put 5-10 small sterile beads on the plate .
11. Pour bacteria on beads and shake plate .
12. Discard beads .
13. Incubate at 37 degree Celsius overnight .
( or 25 degree Celsius for 48 hours )
Pick Colonies ( E.coli )
1. Prepare bacterial culture tubes .
2. Move in hood .
3. Add 2-3 ml LB medium .
4. Use pipette to pick up colony .
5. Disconnect the tip in the bacterial culture tube .
6. Incubate at 37 degree Celsius , shake overnight .
Prepare DNA electrophoresis agar
1. Add 1 g agar
2. Add 100 ml TBE buffer solution .
3. Microwave for 2 minutes .
4. Cool it with water .
5. Add dye . ( invitrogen dye : add 1 ul )
6. Pour it in the casing .
7. Use tip to clean the bubbles .
8. Wait .
9. Store in TBE buffer , at 4 degree Celsius .
Ligation
1. Prepare Eppendorf .
2. Add 2 ul of digestion plasmid .
3. Add equimolar amount of SpeI digested fragment ( < 3 ul )
4. Add equimolar amount of XbaI , PstI digested fragment ( < 3 ul )
( the ratio of plasmid and fragment = 1 : 3 )
5. Add 1 ul ligase buffer .
6. Add 0.5 ul DNA ligase .
7. Ligate at 16 degree Celsius for 30 minutes .
8. Kill the enzyme at 80 degree Celsius for 20 minutes .
9. Transform .
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Miniprep
A. PD1 buffer contained :
Tris buffer
EDTA
Glucose
RNAaseA
B. PD2 buffer contained :
NaOH
SDS (Sodium Dodecyl Sulfate )
C. PD3 buffer contained:
Potassium Acetate
1. Prepare enough eppendorfs .
2. Add bacteria culture .
3. Spin down bacteria . ( 4000 rpm , 5 – 10 minutes )
4. Discard suspension .
5. Vortex pellet to resuspend .
6. Add 200 ul PD1 buffer with RNAaseA .
7. Invert tubes for 10 times .
8. Add 200 ul PD2 buffer .
9. Gently invert tubes for 10 times .
10. 2 minutes at room temperate .
11. Add cold 300 ul PD3 buffer .
12. Gently invert tubes for 10 times .
13. 2 minutes at 4 degree Celsius .
14. Spin down . ( 14000 rpm , 10 minutes )
15. Carefully transfer the suspension to another tube . ( discard the pellet )
16. Add 520 ul cold Celsius isopropanol . ( store at 4 degree Celsius )
17. Invert tubes for 10 times .
18. Spin down . ( 14000 rpm , 5 minutes )
19. Discard suspension .
20. Add 520 cold ethanol . ( store at -20 degree Celsius )
21. Invert tubes for 10 times .
22. 10 minutes at -20 degree Celsius .
23. Spin down . ( 14000 rpm , 5 minutes )
24. Discard suspension .
25. Use pipette to remove suspension completely .
26. Open the lid , and let the pellet dry in the 37 degree Celsius incubator .
27. Add 20 ul double filtrate water .
28. Resuspend DNA in the water .
29. Spin down .
30. Store at -20 degree Celsius .
SDS-PAGE Sample Prepare
1. Prepare Eppendorf
2. Add 200 ul of bacterial culture .
3. Spin down . ( 14000 rpm , 1-2 minutes )
4. Use pipette to remove the suspension .
5. Add 10 ul of sample buffer and 4X dye .
6. Pipetting
7. 5 minutes at 98 degree Celsius .
8. Store at -80 degree Celsius .
( or store at 4 degree Celsius for short time )
PCR
1. Prepare Eppendorf
2. Add 5 ul of 10X standard Taq buffer
3. Add 1ul of 10 mM dNTP .
4. Add 1ul of 10 uM forward primer .
5. Add 1ul of 10 uM reverse primer .
6. Add 0.25 ul of Taq DNA polymerase .
7. Add 5 ul of DNA template . ( can use bacteria culture instead )
8. Add double filtrate water to 50 ul ( 10X buffer )
9. Set PCR 30 cycle
( 98 degree Celsius for 30 seconds , 57 degree Celsius for 30 seconds , 72 degree Celsius for 2 minutes , start at 98 degree Celsius , end at 72 degree Celsius )
Digestion
1. Prepare Eppendorf .
2. Add restriction enzymes . ( each 2 ul )
3. Add 5 ul of DNA
4. Add 5 ul of digestion buffer . ( as known as “cutsmart” )
5. Add double filtrate water to 50 ul .
6. 2 hours at room temperature . ( or overnight )
7. Run DNA agarose electrophoresis .
8. Gel extraction .
Induce protocol for MG1655 and BL21 protein express
1.Transform expression plasmid into BL21. Plate on antibiotic selection plates and incubate overnight at 37°C.
2.Resuspend a single colony in liquid culture with antibiotic to produce a starter culture. Inoculate starter culture at a 2:100 dilution into
3.Incubator at 37°C with shaking until OD600 reaches 0.6–0.8. (approximately 3 hours)
4.Induce with 1mM IPTG and express protein for 3 hours at 12℃, or overnight at 12℃
