Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector pcDNA3.1 digest with...

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Lab meeting 13.03.25

Transcript of Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector pcDNA3.1 digest with...

Page 1: Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector  pcDNA3.1 digest with ScaI- blunt end  Plasmid preparation using Midi kit.

Lab meeting 13.03.25

Page 2: Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector  pcDNA3.1 digest with ScaI- blunt end  Plasmid preparation using Midi kit.

Plasmid preparation and Linearize the pcDNA3.1 vector

pcDNA3.1 digest with ScaI- blunt end

Plasmid preparation using Midi kit from Quiagen

Page 3: Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector  pcDNA3.1 digest with ScaI- blunt end  Plasmid preparation using Midi kit.

Set up protocol to determine Neomycin sensitivi-ties of Hela

300 μg/ml

400 μg/ml

400 μg/ml

500 μg/ml

500 μg/ml

600μg/ml

600 μg/ml

1000 μg/ml

1000 μg/ml

1200 μg/ml

1200 μg/ml

Control Control

split a confluent plate so the cells will be approximately 25% confluent

Neomycin 10mg/ml

A1-2 : 500 μg/ml cells + 0 μg/ml Neo + 500 μg/ml RPMI 10% FBS P.S

A3-4 : 500 μg/ml cells + 30 μg/ml Neo + 470 μg/ml RPMI 10% FBS P.S

A5-6 : 500 μg/ml cells + 40 μg/ml Neo + 460 μg/ml RPMI 10% FBS P.S

B1-2 : 500 μg/ml cells + 50 μg/ml Neo + 450 μg/ml RPMI 10% FBS P.S

B3-4 : 500 μg/ml cells + 60 μg/ml Neo + 440 μg/ml RPMI 10% FBS P.S

B5-6 : 500 μg/ml cells + 100 μg/ml Neo + 400 μg/ml RPMI 10% FBS P.S

C1-2 : 500 μg/ml cells + 120 μg/ml Neo + 380 μg/ml RPMI 10% FBS P.S

Total volume of Hela cells + selective medium : 1 mL

Replenish the selective media every 2-3 days, and observe the percentage of surviv-

ing cells.

Count the number of viable cells at regular intervals to determine the appropriate

concentration of antibiotic that prevents growth within 1–2 weeks after addition of

the antibiotic.

300 μg/ml

Page 4: Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector  pcDNA3.1 digest with ScaI- blunt end  Plasmid preparation using Midi kit.

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Contamination is a problem in cultures

The problem and detection of contamination caused by aggressive cell lines, bacteria, mycoplasma, yeast, virus or fungus

Page 5: Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector  pcDNA3.1 digest with ScaI- blunt end  Plasmid preparation using Midi kit.

What are Mycoplasma?

• Kind of bacteria• Absence of cell wall, flexible membrane easy taking in different shapes

and difficulties in identifying under electron microscope• Small size (0.15-0.3μm) main reason for their escape through filtering

systems and also their growth in high concentration in mammalian cell cultures without any turbidity or other obvious symptoms

Page 6: Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector  pcDNA3.1 digest with ScaI- blunt end  Plasmid preparation using Midi kit.

Inhibition of cell proliferation up to 50% by nutrient withdrawal and secretion of harmful metabolic products

• McGarrity et al. (1984) In Vitro Cell. Dev. Biol. 20:1– fast glucose reduction and formation of acids => pH shift– arginine depletion => inhibition of protein biosynthesis, cell division and growth

Influence of immunological reactions (macrophage activation, inhibition of antigen presentation, in-duction of signal transduction)

• Muhlradt et al. (1996) Biochemistry 35:7781– Influence of virus proliferation and the infection rate

• Nar-Paz et al.(1995) FEMS Microbiol. Lett. 128:63– Cause chromosomal aberrations and multiple translocations

• McGarrity et al. (1978) In: Mycoplasma infection of cell cultures.– Disturbance of the hybridoma technique, contaminated cells become accumulation of my-

coplasma at cells wall alters cell wall integrity

Mycoplasma contamination & Effects on Cell Cultures

Page 7: Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector  pcDNA3.1 digest with ScaI- blunt end  Plasmid preparation using Midi kit.

Effects on Cell Cultures (continued)

Significant changes in gene expression profiles Mycoplasma can constitute up to 50% of the total protein and 15- 30% of

the isolated DNA Decrease of the transfection rates by 5% through electroporation Induction of leopard cells (condensation of the chromatins) Influence almost all functions of the host cell metabolism

Page 8: Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector  pcDNA3.1 digest with ScaI- blunt end  Plasmid preparation using Midi kit.

Important of the Mycoplasma Tests

1) may show as abnormal cell growth , decreased growth rate, reduced saturation.

2) invisible under regular microscopy.

3) Standard antibiotics can allow contamination levels lower than detection levels, Pen/Strep does not provide protection from contamination

4) requires kits for testing.

Page 9: Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector  pcDNA3.1 digest with ScaI- blunt end  Plasmid preparation using Midi kit.

Regulations:• FDA Points to Consider (May 1993), Regularien 21CFR610.30• USDA federal code #9CFR113.28• European Pharmacopoeia 2.6.7, Suppl. 5.8• ICH Guideline for biotechnological/biological products Obliged to test:• Master cell cultures, cell cultures, virus stocks, control cell cultures• Bioproducts from cell cultures (antibodies, hormons, immune stimulators,

blood• products from cell cultures)• Vaccines for humans and the veterinary field Test necessary for:• Editors who are aware of the significance of mycoplasma contamination

Instruction for Testing

Page 10: Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector  pcDNA3.1 digest with ScaI- blunt end  Plasmid preparation using Midi kit.

Detection of Mycoplasma containmination on Hela

• DAPI staining

Page 11: Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector  pcDNA3.1 digest with ScaI- blunt end  Plasmid preparation using Midi kit.

Detection of Mycoplasma containmination on Hela (continued)

• PCR method• e-Myco™ plus Mycoplasma PCR Detection Kit

• Target band: 268 bp ~ 277bp• Inetrnal control 160 bp Positive Negative Hela Ladder

Page 12: Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector  pcDNA3.1 digest with ScaI- blunt end  Plasmid preparation using Midi kit.

Mycoplasma Elimination kit