How do you troubleshoot DNA extraction?
Poor DNA Extraction Proteinase K inactive because it was prepared too far in advance. Prepare Proteinase K within one hour of use. Water baths not at proper temperature Use a thermometer to confirm water bath set point. Not enough DNA Try cheek cell extraction.
What is the purpose of using CTAB during DNA extraction?
One of the most commonly used methods to extract DNA from plants uses the ionic detergent cetyltrimethylammonium bromide (CTAB) to disrupt membranes and a chloroform-isoamyl alcohol mixture that separates contaminants into the organic phase and nucleic acid into the aqueous phase.
Why β mercaptoethanol is used during the preparation of CTAB buffer?
b-Mercaptoethanol is often included in extraction buffers designed for plant DNA extraction, because it is a strong reducing agent which can remove tannins and other polyphenols often present in the crude plant extract.
Do we need to autoclave CTAB buffer?
Preparation for CTAB Add Tris and EDTA in the solution, then autoclave it. Never mind if CTAB and NaCl are not thoroughly dissolved. After autoclaving, the solution will be transparent.
What are the factors that may cause error in DNA extraction?
The two sources of errors which occur during PCR amplification of DNA are (1) mistakes made by the polymerase and (2) thermal damage of the DNA in double-and single-stranded form.
What is the pH of CTAB buffer?
pH 8.0
CTAB extraction buffer
| Reagent | Amount to add (for 10 mL) | Final concentration |
|---|---|---|
| 0.5 M EDTA (pH 8.0) | 0.4 mL | 4% |
| 1 M Tris-Cl (pH 8.0) | 1 mL | 10% |
| Polyvinylpyrrolidone (PVP) (MW 40 kDa) | 0.3 g | 3% |
| β-Mercaptoethanol | 0.02 mL | 0.2% |
What is the principle of CTAB method?
Principle: CTAB (Cetyl trimethyl ammonium bromide) is a detergent used to break open plant cells and solubilize its contents. The extraction process involves breaking or digestion of cell wall in order to release the cellular constituents.
What is the role of beta-mercaptoethanol in DNA extraction?
β-Mercaptoethanol (HOCH2CH2SH) is added most of the time in extraction buffers and is a strong reducing agent to clean tannins and other polyphenols present in the crude plant extract. Globular proteins get dissolved in water.
Is 2-Mercaptoethanol the same as beta-mercaptoethanol?
Gibco™ 2-Mercaptoethanol (also known as beta-mercaptoethanol or BME) is a potent reducing agent used in cell culture media to prevent toxic levels of oxygen radicals. 2-Mercaptoethanol is not stable in solution, so most protocols require daily supplementation.
How do you dissolve a CTAB buffer?
Dissolve 4.1 gram NaCl (Merck, p.a.) in 80 mL distilled water. While stirring, add 10 gram N-cetyl-N,N,N,-trimethyl ammonium bromide (CTAB) (Merck, p.a.). To dissolve heat the solution at 65 °C. Adjust the volume to 100 mL with distilled water.
Does CTAB buffer expire?
Prepare CTAB Buffer (recipe attached). Prior to starting extraction, add polyvinylpyrrolidone and β-mercaptoethanol. Once these have been added the shelf life of the buffer is only 2-3 days.
Why is cold isopropanol used in DNA extraction?
The use of chilled IPA increases the rate of precipitation of DNA and allows it to flocculate and settle very easily and quickly. However, its recommended to use room Temperature Isopropanol because cold Isopropanol may increase precipitation of salt.
What factors could affect the success of the extraction of DNA?
What factors could affect the success of extraction of DNA from strawberries or other plants? Answers may include: amount of DNA in a particular fruit, the number of cells in the fruit, ripeness of the fruit, following procedures, etc.
What errors could lead to not having a band on the gel after electrophoresis?
If you see faint or no bands on the gel: There was insufficient quantity or concentration of DNA loaded on the gel. Increase the amount of DNA, but don’t exceed 50 ng/band. The DNA was degraded. Avoid nuclease contamination.
What are common errors when doing gel electrophoresis?
Sample Leaking Out of Well. Bands Are Smeared Vertically. Too Many Bands. Gel Running Unusually Slowly.
What is the CTAB buffer?
CTAB Extraction Buffer is a widely-used reagent used to isolate DNA from plant tissues. Polysaccharides and polyphenols are problematic contaminants associated with DNA isolated from plants.
Is 2 mercaptoethanol the same as beta-mercaptoethanol?
How bad is beta-mercaptoethanol?
BME can be toxic if ingested, and fatal if inhaled or absorbed through the skin. Vapors can irritate the eyes, mucous membranes, and respiratory tract. Symptoms of inhalation exposure may include coughing, sore throat, and/or shortness of breath.