PH Meter using protocol

P^H Meter using protocol

1.     Make available 3 A water in rinsing bottle, paper towel, a beaker and buffer solution (P^H 7.00 and P^H 9.21)

2.     Turn on the machine.

3.     Uncap the probe and rinse it with 3A water.

4.     Dry the probe by using paper towel but not keep it dry for long time because it will damage the probe.

5.     Submerge the probe in buffer (P^H 7.00) immediately. Press the calibration Button.

6.     Take out the probe from buffer (P^H 7.00) and rinse it with 3A water.

7.     Dry the probe with paper towel and submerge the probe in buffer (P^H 9.21) asp.

8.     Press the calibration Button and read the slope. Write down the slope on record book.

9.     Take out the probe from buffer (P^H 9.21) and rinse it with 3A water.

10.    Dry the probe with paper towel and submerge the probe in target liquid if it is not ready put it 3A water and ready the target liquid. When the target liquid is ready, take out the probe and dry it with paper towel and submerge it in target liquid.

11.    Press the Read Button and read the P^H

12.    If require adjust the P^H by using 10N NaOH and con. HCl (Wear gloves before  using them)

Protocol for Raw 264.7 Cell Thawing

Protocol for Raw 264.7 Cell Thawing

1.     Warm water bath to 37 °C.

2.     Place 10 mL media DMEM in a sterile 15 mL centrifuge tube.

3.     Make your growth media (DMEM) for your new culture (10 ml DMEM in a 75 cm² Tissue culture flask)

4.     Take your cells out of nitrogen storage and thaw rapidly by swirling in the 37 °C water bath.

5.     Sterilize the outside of the vial with 70% ethanol, bring in to the culture hood and add the cells slowly to the media you prepared.

6.     Centrifuge at 1200 rpm, room temperature for 5 minute.

7.     Pipet off the supernatant. Then, add DMEM and pipet up and down to resuspend them in DMEM.

8.     Pipet the cells into a 75 cm² Tissue culture flask containing 10 ml DMEM and incubate at 37⁰C, 5% CO2 in an incubator for growth of your new culture.

9.     After overnight incubation pipet off the old media and add new media. The incubate again at 37⁰C, 5% CO2 in an incubator.

Protocol for Middlebrook 7H9 Broth (Difco) with Glycerol

Protocol for Middlebrook 7H9 Broth (Difco) with Glycerol

Material: Middlebrook 7H9 Broth Powder, Glycerol, 20% Tween 80, OADC

1.     Take 900 ml distilled water in a glass flask

2.     Place the flask on a magnetic stirrer

3.     Add 4.7 g of the powder in water and stir it

4.     Add 2 ml of glycerol (Pipette slowly) and stir it

5.     Autoclave the medium at 121⁰C for 15 minutes

6.     After Autoclaving cool the medium up to 45⁰C

7.     Then, aseptically add 100 ml of OADC to the medium*

8.     Add 2.5 ml of 20% Tween 80 to the medium *

9.     7H9 broth is ready, store it at 4⁰C

* Steps 7 and 8 must be done in a safety hood

* Temperature higher than 50⁰C can denature OADC and Tween 80.