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Team:Potsdam Bioware/Lab/Protocols
From 2012.igem.org
Protocols
Media:UP12_Dephosphorylation.pdf
Media:UP12_Gelelectrophoresis.pdf
Media:UP12_Mutagenesis-PCR.pdf
Media:UP12_Preparative digestion.pdf
Media:UP12_Transfection protocol CHO.pdf
Media:UP12_CHO - Transfektion.pdf
Other protocols
Fluorescent protein purification
1. Prepare 50ml of LB medium with appropriate antibiotics, inoculate with glycerol stock of the strain of interest (E.coli XL 1 Blue with CFP/YFP)
2. Nurture overnight at 37°C, 200rpm.
3. Inoculate 10ml of the overnight culture into 2x 500ml LB medium with antibiotics
4. When OD600 is 0,3-0,5 add IPTG to the final concentration of 500 mM.
5. Nurture for another 6 hours and centrifuge (Beckman centrifuge: 5000rpm, 4°C, 45min)
6. Freeze in -80°C freezer
7. Resuspend the pellet in 20ml HEPES buffer
8. Transfer to 50ml Falcon tubes
9. Sonication (3x2min) on ice – 6mm tip, cycle 50%, output 5-6, timer HOLD.
10. Centrifugation: 30 min, 19000rpm, 4°C
11. Transfer the supernatant to new Falcons
12. Sonicate for 1min
13. Filter through 0,45µm filter
14. Store on ice before loading at the column
15. Equilibrate the column: 30 times column size with HEPES buffer
16. Load the column with the sample
17. Wash with washing buffer
18. Elute with elution buffer
19. Load the protein gel
HEPES buffer:
10mM HEPES
500mM NaCl
Washing buffer:
10mM HEPES
500mM NaCl
30mM Imidazol
Elution buffer:
10mM HEPES
500mM NaCl
50mM Imidazol
Phage amplification and clean up:
1.Overnight culture of 20ml LB+Tet+ER2738 stock solution
2. Main culture on the next day (200ml LB+tet+overnight culture)
3. Addition of phage when OD600 0,3-0,5
4. After addition of phage: 15min in 37°C (no shaking)
5. Move to 28°C incubator
6. After 45min add Kana
7. After 4h (divide into 4x50ml Falcon tubes)
8. centrifuge 5000g, 15min, 4°C
9. Supernatant into new tube
10. centrifuge 5000g, 15min, 4°C
11. 4x40ml of supernatant + 4x8ml PEG-NaCl solution
12. Incubate on ice overnight in the fridge
13. Centrifuge: 5000g, 45min, 4°C
14. Discard supernatant
15. Centrifuge 5000g, 5min, 4°C
16. Remove supernatant with pipette
17. Resuspend pellet in 4x1ml TBS (pH 7,5)
18. Transfer to 4x 1,5ml Eppie
19. Centrifuge 21000g, 10min, 4°C
20. 4x900µl of supernatant mix thoroughly with 4x180µl of PEG-NaCl solution
21. Incubate on ice for 1h
22. Centrifuge 21000g, 10min, 4°C
23. Remove supernatant with pipette
24. Resuspend pellet in 0,3ml TBS (pH 7,5)
25. Centrifuge 21000g, 10min, 4°C
26. transfer 280µl of supernatant into new 1,5ml Eppie
PEG-NaCl (100ml):
20% w/v polyethyleneglycol 6000-20g
2,5M NaCl in water
TBS:
50mM Tris
150mM NaCl
pH 7,5 HCl/NaOH
RNA-Extraction with Phenol/Chloroform and LiCl-Precipitation from eukayrotic cells
2x Lysis Buffer
1 M Tris-Cl, pH 7.5 2 mL
5 M NaCl 1,2 mL
0,5 M EDTA pH 8 1,2 mL
H2O 7,2 mL
10% SDS 8,0 mL*
- Note
Add in order given, otherwise SDS will precipitate. Don’t put on Ice afterwards, otherwise SDS will precipitate.
Procedure
1) Use bottles or tubes that are set aside for RNA work. Harvest 15ml cells by centrifugation for approx. 2 minutes @ 2500g. Cells should make a nice pellet.
2) Discard the supernatant and take up the all liquid with a pipette.
3) Resuspend cells in 600 µl 1x Lysis Buffer to cell suspension and shake slowly for 20 minutes at 4°C in coldroom.
4) Set up 2ml tubes for phenol/CHCl3 extractions
5) Extract cell suspension with 600µl ml Phenol/CHCl3/Isoamyl Alcohol (25:24:1, e.g. Roti© from ROTH) and shake well for 1 minute
6) Centrifuge @ 12000g for 5 minutes.
7) Collect upper, aqueous phase (avoid interphase material) and repeat steps 5-7. (Total of 4 Phenol/CHCl3 extractions. Do not worry if phase is pink.)
8) After second extraction add 6µl of 4mg/ml proteinase K solution (Roche) and incubate for 10 min at 65°C.
9) Repeat the steps 6-8 twice.
10) Transfer the upper, aqueous phase to a new tube.
11) Extract the upper with 400µl CHCl3/Isoamyl Alcohol to reduce phenol concentration.
12) Centrifuge 2,5 minutes @ 12000g.
13) Take upper, aqueous phase and transfer to a new 1,5ml tube.
14) Add same volume LiCl (5M) to a total concentration of 2,5M.
15) Incubate for 1h at -20°C.
16) Centrifuge @ 16000g for 20min at 4°C.
17) Discard supernatant, be carefull with the pellet.
18) Perform ethanol-precipitation: Add 500µl of 100% Ethanol to the pellet an resolve it.
19) Incubate the tube for 30 min at -20°C.
20) Centrifuge @ 16000g for 20 min at 4°C.
21) Wash pellet in 70% cold ethanol and dry in speed vac.
22) Resuspend pellet in 40 µl H20. Store at -80°C.
Glycerol Stocks of transformed Cells
- before minipreping
take 500 µl E.coli culture add 500 µl 99.8% Glycerol
freeze at -80°C
Recipes for stock solutions
Ampicillin (Amp)
Stock solution: 100 mg/ml
- Filter in H2O (Milipore) !
Working concentration: 20-100 μg/ml (50 μg/ml)
Chloramphenicol (Cm)
Stock solution: 25 mg/ml
- In 70% EtOH
Working concentration: 25-170 μg/ml (100 μg/ml)
Kanamycin (Kan)
Stock solution: 50 mg/ml
- Filter in H2O (Milipore) !
Working concentration: 10-50 μg/ml (30 μg/ml)
Tetracycline (Tet)
Stock solution: 25 mg/ml
- In 70% EtOH
Working concentration: 10-50 μg/ml (10 μg/ml in liquid culture; 12,5 μg/ml in Plates )
- Be careful: Light sensitive, take aluminium foil and wrap tube!
TAE (50x)
1L TAE (50x)
242 g tris base
57.1 ml glacial acetic acid
100 ml 0.5 M EDTA (or 18.612 g EDTA Disodiumsalt dihydrate M=372.24)
ad 1 L
CaCl2
1 M CaCl2
- 110,98 g CaCl2
- 1 l H2O milipore
1l
CaCl2 x 2 H2O : M= 147,02 g/mol
- weight 147,02 g
- fill up to 1000ml with Milipore H2O
- steril filtration (Big Filtration)
DYT
DYT growing medium
5 Liter:
80g Bactotrypton
50g Bacto yeast
25g NaCl
- add 2l Multipore –H2O
- add the magnetic stir bar
- ad 5l Multipore –H2O
- fill into a jar
- autoclave
LB-Medium + Agar
Preparation of LB-Medium + Agar:
Only start to prepare the LB Medium if it will be autoclaved on the same day (8:00 am, 10:00 am, 12:00 am, 14:00 pm).
Always work with a measuring cylinder !
Take a 1L flask and fill in:
- 10 g Bactotryptone
- 10 g NaCl
- 5 g Bactoyeast
- Add 500 ml Millipore H2O and dissolve the three substances.
- While dissolving, prepare 19 or 20 100 ml flasks and fill in 0,75 g Agar (0,015 g Agar/ml).
- Label it with your name, the date and LB+Agar.
- As soon as the substances are dissolved in 500 ml millipore H2O add another 500 ml.
- Then fill 50 ml LB-medium in each 100 ml flask and autoclave it.
Competent E.coli cells
Production of competent E-coli-cells
Work always sterile and cold and speedy!
- All volumes deal with the common cell line!
- The cooling-centrifuge is in the tool shed , cool down to 4°C early enough, close the lid correctly!
- Prepare early enough min. 100 Eppis (1,5μl) (per cellline) and cool down to -80°C before using
- Use Milipore-filter for sterile CaCl2 , keep cool!
- prepare 15ml LB-Medium (or DYT) with the specific antibiotic (XL1-blue Tet, BL21 none!), inoculate and incubate over night
- prepare 200ml LB-Medium (or DYT) with the specific antibiotic, inoculate with 2ml of the over-night-culture. Nurture the culture until OD600 at 0,35 (0,2-0,5) (if the OD is too high, the cell won’t be competent)
- keep cell suspension in sterile falcons (50ml) 20 min on ice, then centrifuge for 20min; 4°C; 2500g
- discard supernatant, carefully resuspend on ice with 10ml cold CaCl2-solution (put a little of the 10ml solution in every falcon before!), pool every resuspended aliquot of one cell line and add 10ml CaCl2-solution (total volume 20ml). Keep 30 min on ice, then centrifuge for 20min; 4°C; 2500g
- discard supernatant, carefully resuspend pellet in 5,5ml CaCl2(80mM)/Glycerol (4:1), aliquot in Eppis á 60μl and store immediately at - 80 °C
Glycerol
- fill Glycerol into a flask
- autoclave
IPTG
MW= 238,3 g/mol
1M Stock solution: 238,3g in 1l H2O
- 20ml: 4,766g in 20ml H2O
- filtern (“sterilize by filtration)
- this solution is stable for 2-4 months
TE Buffer (1×)
100ml
10 mM Tris-HCl (pH 7.5) -> M= 121,14 g/m
1 mM EDTA -> M=egal; c=0,5M
Autoclave
- 1,2114g Tris + 0,2 ml EDTA (0,5M)
- add 70 ml H2O (Milipore)
- titrate with HCl to pH 7,50
- ad 100 ml H2O (Milipore)
- filter
Check for competent Cells
- PUC18 50pg/μl -> 2 μl to 1 Eppi of cells
- Trafo
- Amp
- 1Cana +1Amp –additional plate with untransfected cells for control
Make DNA Ladder Mix (Gene Ruler, Thermo Scientific) ready to use
- if you work with Gelred DON`T follow the [http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_sm0331.pdf instructions from Thermo Scientific]! DNA-Ladder must be diluted 10x stronger because Gelred is very sensitiv. In the end the DNA Ladder must be diluted 1:60 in 1x Loading Dye Solution.
Example: Mix 10 µl DNA Ladder, 120 µl 5x Loading Dye Solution and 470 µl deionized wather --> 600 µl Ladder ready to use
