How much DEPC do you add to water?

How much DEPC do you add to water?

Mix 1 mL of DEPC (diethyl pyrocarbonate) with 1000 mL of water in a screw-cap glass bottle. Incubate for ~2 h at room temperature in a fume hood with occasional swirling. Autoclave. Store at room temperature for up to 12 mo.

What does DEPC water do in PCR?

DEPC-treated Water – DEPC destroys enzymatic activity by modifying -NH2, -SH and -OH groups in RNases and other proteins. DEPC treatment is a very effective way to treat solutions that will contact RNA.

Why is DEPC treated water not recommended for PCR?

DEPC is not recommended as it can be slightly acidic, which can lead to depurination and degradation of the oligos.

How do you dissolve DEPC in water?

DEPC Treated Water Recipe

  1. Add 1ml of 0.1% Diethylpyrocarbonate (DEPC) to 1000ml distilled water.
  2. Mix well and let set at room temperature for 1 hour.
  3. Autoclave.
  4. Let cool to room temperature prior to use.

Is DEPC water Light Sensitive?

Caution, DEPC is irritating to eyes, skin and mucus membranes. It is a suspected carcinogen. Light sensitive. Open DEPC only in fume hood, wear gloves.

How do you prepare DEPC water or nuclease free water?

Popular Answers (1)

  1. Get MilliQ (reverse osmosis purified) water.
  2. Add 1 ml DEPC (Diethylpyrocarbonate) per 1000 ml of MilliQ or double distilled water (i.e. to a final concentration 0.1%) and mix thoroughly.
  3. Let the DEPC-mixed water incubate for 12 hours at 37°C.
  4. Autoclave DEPC-mixed water for 15 minutes.

Is DEPC water deionized?

Thermo Scientific deionized, diethylpyrocarbonate (DEPC) treated and 0.22 µm membrane-filtered water. It is ideal for applications involving RNA. Free of endo-, exodeoxyribonucleases, ribonucleases, and phosphatases.

What is the pH of DEPC water?

around 3.97-.4.08
But what i have observed many times is that the pH of the autoclaved DEPC treated water is around 3.97-. 4.08.

Is DEPC treated water toxic?

1. DEPC is toxic and not appropriate in a lab where it cannot be handled properly.

Is DEPC toxic?

DIETHYLPYROCARBONATE (DEPC) ( \f )1609-47-8 Harmful if swallowed. Symptoms/injuries after skin contact : Tingling/irritation of the skin.

How do you store DEPC?

After opening the bottle for the first time and each time thereafter, layer nitrogen or argon gas over the DEPC and store the closed bottle at 2–8 °C for optimal stability. It may be helpful to store the bottle inside a sealed plastic bag with desiccant, but with the bottle cap slightly loose.

Why is nuclease-free water used?

The presence of nucleases such as DNase and RNase in water can degrade precious molecular samples and even ruin experiments. To prevent DNA and RNA sample loss, it is essential that highly pure, nuclease-free water be used in applications such as PCR, cDNA synthesis, nucleic acid purification, sequencing, and cloning.

Is DEPC carcinogenic?

DEPC is a potent, non-specific chemical inhibitor of RNase2 and its most common application has been to make water nuclease-free. DEPC is also carcinogenic and should be handled with care and with strict attention to the safety recommendations of the manufacturer.

Does DEPC expire?

Once opened, the bottle should not be kept for more than 9 months. 2. To decontaminate glassware of RNase, the glassware may be incubated with a DEPC solution for 30-60 minutes, and subsequently autoclaved to decompose the DEPC.

What is the difference between Milli Q water and distilled water?

Milli-Q water is produced by reversed osmosis that normally can even remove bigger ions. Distilled water is purified via distillation i.e boiling and then condensation of the water. So, a double distilled water has gone through the process of distillation twice.

Why do we need nuclease?

Nuclease free water is used in order to dilute the concentration of the reagents to the proper final concentration. Also use of nuclease free water helps avoid DNA degradation by nucleases as well as interference of the PCR reaction by ions which could be present in otherwise not nuclease free deionized water.

What is DEPC-treated water used for?

It has also been used as a probe for the topography of 5.8S rRNA yeast ribosomes. DEPC-Treated Water is suitable for use with RNA. It is prepared by incubating 0.1% DEPC and is then autoclaved to remove the DEPC.

What is the effect of DEPC on ultrapure water?

Treating ultrapure water with DEPC inactivates RNAse which can degrade RNA in valuable samples. After DEPC treatment, the water is autoclaved to eliminate the bi-products of the DEPC treatment.

How do you get rid of DEPC in water?

Water is usually treated with 0.1% v/v DEPC for at least 2 hours at 37 °C and then autoclaved (at least 15 min) to inactivate traces of DEPC. Inactivation of DEPC in this manner yields CO 2 and ethanol.

What happens to the water after DEPC?

After DEPC treatment, the water is autoclaved to eliminate the bi-products of the DEPC treatment. If you’re using specific enzymes or reverse transcriptase, you require water that doesn’t contain RNAse which can interfere with RNA that may be the focus of your research. Sterility of water is measured by the Sterility Assurance Level (SAL).