1-) Wash the cells in the flask with 5 mL 1X PBS twice. At this stage removing PBS completely is very important. Otherwise activity of Trypsin decreases

2-) Add 2 mL Trypsin on cells. Be careful; Trypsin should be spreaded whole surface. Then wait 5 minute at 37&;°CBecause trypsin works maximum activity at 37 ° C.

3-) After we sure that cells remove from surface completely, wash the surface carefully with 5 mL medium via pipetting .

4-) Take all the mixture which we prepared to falcon tubes( 15 mL) and centrifuge 6 minutes.

5-) Supernatant should be taken carefully without damaging to cells.

6-) Add 5 mL medium on pellet and dissolve the pellet in medium.

7-) Take 100 mL from cell mixture, take 100 mL from Tripan-Blue and mix in eppendorf.

8-) Take 20 micro liters to Hemocytometer( Thoma lam) and count after then.

9-) We will put the lam to microscope. Lam’s H zone should be under the light.

10-) There are 5 areas that should be counted.

11-) Total number of cells which at 5 areas, are counted at arithmetic average is counted.

12-) To count the total cell number;

*Cell number: Average cell count x dilution factor x

*Dilution Factor: It is the rate between dye and cell contents. For this protocol dilution factor is 2.(1+1)

*For example; 68x2x=1.360.000(cell count per mL). But we want to seed 500.000 cell per mL.

1 mL 1.3 million

X 0.5 million

X= 0,385 mL = 385 microliters

*So we will take 0.385 microliters to seed 500.000 cells.

*According to the material ( where will we seed our cells? Flask, petri, well..) amount of medium is counted and the cells are seeded.

*For example, according to six well-plate; 2 mL medium is put, Than 385 microlitres cells are added.

*For six well-plate, the best situation is adding 500.000 cells.

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1 -) The medium stored at +4 ° C, is heated at 37 ° C at least 30 minutes.

2 -) Remove all mediums from the flasks carefully without damaging the cells. At this stage, the medium is in the flask, can be poured directly or can be taken with pipette.

3 -) 10 mL medium is taken which heated and clean, is added to in the middle of flask. This stage is done very slowly and absolutely avoiding the cells. After than flask is careened slowly (medium should not touch to flask’s cap) to mix the cells with medium.

4-) While changing medium, we must be careful and fast as much as we can.

*The amount of solution according to flask type;

1 -) Set Benmari at 37 ° C.

2 -) The cell line stored at -80 ° C, is brought to cell culture room in ice. Then put in 37 °C and wait 2 minutes.

3 -) After melting cells for 2 minutes, cryovial tubes are cleaned with alcohol and their caps are opened.

4 -) All the stock cells are put in to 5 mL medium placed 15 mL falcon tubes.

5 -)Do pipetting the Medium-Cell mixture carefully. At this stage, it is important not to harm the cells. So don’t work fast, don’t hurry. Work slowly and gentle.

6 -) Put 9 mL medium is to 75 mL empty flask.

7 -) Add the mixture, which was prepared in falcon tube, to flask. Shake gently to the left and the right way up and down carefully. Take care to never touch the mixture to flask’s cap.

8 -) Put the cells into the incubator and set at 37 ° C and %5 CO2.

The amounts of solution according to flask type

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1. Pick a single colony into 5ul of NFW. (Fresh colonies grown that day work best, but they can also come from 4 C).
2. Boil for 5 min at 95C.

PCR Reaction

Keep all the reagents at 4C while preparing the mixture. Pre-heat the thermocycler to 95C and transfer your reaction directly from 4 C.

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For E.coli(DH-5α)

1-) Streak DH5α directly from a frozen stock onto LB agar plate (Amp-).

2-) Incubate at 37℃ for 12 h.

3-) Inoculate one well isolated colony into 5ml of LB(Amp-).

4-) Incubate at 37℃ until OD₆₀₀ =0.8

5-) Transfer the preculture 5ml to 250ml of LB(Amp-)

6-) Incubate at 23℃ until OD₆₀₀ =0.4-0.5 (120rpm/min )

7-) Place sample on ice for 10min

8-) Transfer the culture to steril ice cold tube(250ml)

9-) 3.5Krpm at 4℃ for 5min

10-) Remove the sup well

11-) Resuspend the pellet by gently vortexing and pippeting in 100 ml of ice cold TB*

12-) Sit on ice for 10min

13-) 3K rpm at 4℃for 5min

14-) Remove the sup well

15-) Resuspend the pellet gently in 25ml of TB

16-) Add 875μl DMSO with disposable pippet

17-) Mix gently by swirling

18-) Sit on ice for 10min

19-) Add 875μl DMSO

20-) Mix gently by swirling

21-) Sit on ice for a few minuites

22-) Dispense the sample into 1.5ml tube sterilized with UV for 10min

23-) Chilled in liquid N2

[Preparation TB]

1-) Add the following into 475 ml milli-Q

2-) PIPES 1.5g

3-) CaCl2･2H2O 1.1g

4-) KCl 9.3g

5-) Adjust pH to 6.7 with 1N KOH

6-) Add 5.45g of MnCl2･4H2O

7-) Add milli-Q upto 500ml

8-) Filtration

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