How much RNase A to add to P1?

How much RNase A to add to P1?

Add the provided RNase A solution to Buffer P1 before use. Use 1 vial RNase A (centrifuge briefly before use) per bottle Buffer P1 for a final concentration of 100 μg/ml. Mix and store at 2–8°C. Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).

What is a miniprep kit?

The Monarch Plasmid Miniprep Kit is a rapid and reliable method for the purification of high quality plasmid DNA. Elution in as little as 30 μl provides concentrated DNA for use in downstream applications, such as restriction digests, DNA sequencing, PCR and other enzymatic manipulations.

How much RNase A to add?

1 to 100 µg/mL
Recommended concentration of RNase A is 1 to 100 µg/mL depending on the application. The enzyme is active under a wide range of reaction conditions. At low salt concentrations (0 to 100 mM NaCl), RNase A cleaves single-stranded and double-stranded RNA as well the RNA strand in RNA-DNA hybrids.

Where does DNA go after miniprep?

Storing DNA: temperature and longevity The most common solution is to keep your plasmid at -20°C or even at -80°C, in this case your preparation can be eluted in water or in your buffer of preference, and it will be stable for years.

What is alkaline lysis method?

Alkaline lysis is the method of choice for isolating circular plasmid DNA, or even RNA, from bacterial cells. It is probably one of the most generally useful techniques because it is a fast, reliable and relatively clean way to obtain DNA from cells.

How do I prepare 10mg mL RNase?

To prepare a 10 mg/mL RNase A stock solution, dissolve 100 mg of RNase A in 10 mL of Tris-Cl (10 mM, pH 7.5)/NaCl (15 mM). Heat to 100ºC for 5 min and cool at room temperature. Store at −20ºC.

How do you use RNase?

To remove RNA from your samples, add RNase, DNase-free and incubate at either +15 to +25 °C or +37 °C. For example, add 0.5 μL RNase to the nucleic acids from 106 cells and incubate at +15 to + 25 °C or +37 °C. For nucleic acids from 107 cells, add 1.5 μL RNase and incubate 30 min at + 37 °C.

What is plasmid miniprep?

Plasmid Miniprep Course Home Page| Laboratory\rHome Page| Laboratory Schedule This procedure is used to extract plasmid DNA from bacterial cell suspensions\rand is based on the alkaline lysis procedure developed by Birnboim and\rDoly (Nucleic Acids Research 7:1513, 1979).

What is MiniMini-prep procedure?

Mini-Prep procedure is used to isolate small plasmid DNA from bacteria while limiting contaminating proteins and genomic DNA. The plasmid quality is acceptable for restriction analysis, sequencing, cloning, or other purposes, but should not be used with out additional cleanup for embryonic injections.

What happens if my DNA Prep is contaminated with a nuclease?

If your DNA prep becomes contaminated with a nuclease (like the ones produced\rby the cells in your skin) then the nuclease will be inactivated by the\rfact that the magnesium cofactor is unavailable in the solution (because\rit is chelated by the EDTA). 19. Pool the two 20 uL solutions into one labeled tube and store\rit in the freezer.

How do I prepare preps for use in a microcentrifuge?

For cleanest preps avoid transferring any precipitate. Microcentrifuge on high (14,000 rpm) for 10 minutes, remove supernatant to new microcentrifuge tubes. ** For cleanest preps avoid transferring any precipitate. Add 1mL of isopropanol to each tube (fill microcentrifuge tube).