Biomod/2012/Potsdam/DnanoPROT/Lab

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Contents

Protocols

Preparation of LB-Medium + Agar

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.

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

Ampicillin (Amp)

Stock solution: 100 mg/ml

  • Filter in H2O (Milipore) !

Working concentration: 20-100 μg/ml (50 μg/ml)

polymerase chain reaction

Mastermix

reagent volume [µL] Final conc.
HF Phusion buffer 5x 10 1x
dNTPs 1 200 μM each
Primer (Forward) 2 0.5 μM
Primer (Reverse) 2 0.5 μM
DNA (Plasmid) 1.0 to 200 ng
Phusion Polymerase 0.5 0.02U/μl
water add to 50


  • add Phusion DNA Polymerase as the last component to the PCR mixture
  • mix and centrifuge all tubes


Cycling conditions

step Temperature [°C] duration [s] cycles
denaturation 98 30 1
denaturation 98 10 30
annealing 45-72 40 30
elongation 72 40 30
final elongation 72 300 1
cooling 4 1

preparative digestion

  • warm up heat block to 37 °C
  • pipette all reagents in a 1.5 ml tube


  • incubation of prep. digestion: 3-4 h at 37 °C

Sticky-end Ligation

temper heatblock to 22 °C
pipette all reagents in a 1.5 ml tube


incubation of sample 1.5 h at 22°C
store ligated samples in a -20 °C freezer

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


Transformation protocol

1. warm up heat block to 42 °C
2. competent cells from -80 °C freezer
3. XL1-blue cells for all cloning + DNA-related; BL21 cells for protein expression (aliquots of competent cells in 2 ml tubes; ca. 50 μl)
4. keep competent cells 15 min on ice
5. add DNA (depending on concentration) into cell-suspension; 0.5 μl for retransformation; 10 μl for transformation
6. incubation on ice ca. 30 min
7. heat shock for 45 sec
8. put tubes back on ice for 5 min
9. add 850 μl pre-warmed (37 °C) LB-medium (antibiotic-free)
10. incubate cells 1 h at 37 °C and 900 rpm (depending on shaker)
11. centrifuge cells for 2 min at 2500 rcf
12. decant supernatant; ca. 50 μl remain in tube
13. resuspend cell pellet
14. plate 20 μl supernatant on LB-plates with adequate antibiotics
15. over-night incubation at 37 °C in incubator
16. pick colonies after 18 – 24 h

Info to: XL1-blue cells (E.coli): The XL1-Blue strain allows blue-white color screening for recombinant plasmids and is an excellent host strain for routine cloning applications using plasmid or lambda vectors. XL-1 cells are tetracycline resistant. XL1-Blue cells are endonuclease (endA) deficient, which greatly improves the quality of Miniprep DNA, and are recombination (recA) deficient, improving insert stability. The hsdR mutation prevents the cleavage of cloned DNA by the EcoK endonuclease system.

Picking clones

1.pick clones from plate
2.inoculate clone in 5 ml LB-medium
3.over-night incubation at 37 °C

Plasmid Miniprep

1. execute miniprep corresponding to manufacturer’s instructions
2. accurate labeling of plasmids
3. measure DNA concentration using nanodrop
4. store plasmids at -20 °C

Gel-electrophoresis

1% Agarosegel

wear Nitril-gloves

  • 0,1 g agarose per 10 ml TAE-buffer (1x) (roughly 60 mL)
  • prepare shot sleeve use matching combs (volume of slot!)
  • liquefy mix for 2 min at 495 W in microwave, boil twice (beware of boiling retardation, heat until no unsolved agarose-particles are visible anymore)
  • add 4 µl EtBr/Gelred depending on gelsize and concentration of EtBr/Gelred

-> caution: EtBr/Gelred evaporates !

  • cast whole gel in chamber (immediate cleaning avoids with water)
  • put combs in liquid gel and let the gel cool down
  • loading probes (digestion): 25 µL DNA-sample (use big chambers)
  • separate at 70 V for 60-70 Min

Gel extraction

1. put gel on UV-table/ -chamber
2. cut desired bonds using a scalpel and transfer the DNA in a 1.5 ml tube
3. extract DNA using gelextraction-kit (follow manufacturer’s instructions)
4. measure DNA concentration using nanodrop

Glycerol Stocks of transformed Cells

before minipreping

take 500 µl E.coli culture

add 500 µl 99.8%

Glycerol freeze at -80°C

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


Protein Expression

1. Inoculation of 25 ml of overnight culture (XL1-blue host strain transformed with EGFR third domain gene and PelB sequece in pBAD) into 1 L of LB medium with ampicillin.

2. Start the expression with 0,05% arabinose solution, when OD600 is at 0.3-0.5

3. 4 hours incubation at 37 °C

4. Centrifugation of cell suspension for 3 min 16100 xg

5. Resuspend pellet in 100µl of lyase sample buffer 4x

6. Pellet is stored at -20 °C

Click-Chemistry Labeling of DNA

1. dilute the peptide sample to a 0,5mg/ml solution in PBS

2. add 500 μL of the sample to an Eppendorf tube

3. add 90 μL of oligonucleotid:DMSO (5 μL : 95 μL ), vortex and leave at room temperature for 1h

4. add 11,6 μL TCEP ( 3,6 mg/ml ), without vortex

5. add 35 μL of TBTA ( 0,24mg/ml in 4:1 tBuOH:DMSO ), vortex

6. add 11,6 μL of copper(II) sulfate ( 4,35 mg/ml ), vortex and leave at room temperature for 1h ( after 30 min vortex )

7. centrifuge for 4 min at 6500g at 4°C

8. add 500 μL cold Methanol rotate and sonicate for 3-4 s.

9. centrifuge for 4 min at 6500g at 4°C

10. add 1 ml 0,4% SDS/PBS, sonicate for 3-4 s

11. heat to 80°C for 5 min

12. add 0,2% SDS with 5 ml PBS

13. samples can be stored at -80°C for several days

DNA origami

1. Mix 40.2 µL of Oligo-pool, 5.661 µL M13 phage DNA, 10 µL 10x HEPES/MgCl2 buffer and 35 µL water.

2. Incubate the mixture for 3 min at 95 °C. After that, cool it with - 1 °C for every min. Stop at 6 °C and hold it.

3. For purification, put DNA origami in millipore (100 kDa) with 400 µL 1x HEPES/MgCl2 and centrifuge it at 4 °C and 5.000 rpm for 3 min. Repeat this step 3 times.

for coupling:

( 20:1 anti-biotin antibody:Origami )

4. Mix 1 μL anti-biotin antibody, 15,56 μL origami and 384 μL HEPES/MgCl2

(2:1 anti-biotin antibody:Origami )

5. Mix 3,15 μL of HEPES/MgCl2:anti-biotin antibody ( 50:1 ), 15,56 μL origami, 21,3 μL HEPES/MgCl2

6. For purification, put DNA origami with in millipore (100 kDa) with 400 µL 1x HEPES/MgCl2 and centrifuge it at 4 °C and 5.000 rpm for 3 min. Repeat this step 3 times.

Safety

The current project and further project ideas of the Potsdam Biomod Team 2012 are classified as biosafety level one (S1) work. None of the ideas do raise additional safety issues to the public, the environment or researchers. We comply with safety regulations given by the law and its amendments, including but not limited to handling and storage of genetically modified organisms and the documentation of the work. The University of Potsdam supervises biological and chemical laboratories and works according to federal and state regulations. A local biosafety officer has been appointed for the lab and the project. About twice a year, the laboratory is physically inspected by the state Authority for Biotechnological Laboratories to ensure compliance with the law and regulations. In Germany, the work with genetically modified organisms is regulated by the ‘Law on Genetic Engineering’ (Gesetz zur Regelung der Gentechnik, GenTG) and amendments of the ”Gentechnik-Sicherheitsverordnung (GenTSV)”, the “Gentechnik-Verfahrensverordnung (GenTVfV)” and the “Gentechnik-Aufzeichnungsverordnung (GenTAufzV)”.

Following federal law, the university provides standard operating instructions for biological & chemical laboratories. All employees and students are instructed before starting to work and attendance is documented by a signature. Rules and instructions are controlled by a safety officer for 'work-, environment- and fire-safety', who is located on our campus.
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