dn4 and rn4 extractioN

eukaryotes, they are found in the nucleus and for prokaryotes, they are found inside the cell as they don't have nucleus.

Where do we find the cell in eukaryotes? And in prokaryotes?

chromatins

Nucleus is organized into what we call as chromosome but before they are chromosome, they are first___

1. Lysis

2. Protein removal

3. DNA Binding

4. Wash

5. Elution

Steps of DNA extraction

Lysis

• Break down of membranes.

• most important part of DNA extraction.

• it releases many components from inside the cell.

DNA column kits

DNA binds to the material that is found in this column.

o NAOH

o SDS (sodium dodecyl sulfate)

o 6M Guanine HCL

Lysis solutions:

6M Guanine HCL

most effective lysis agent as we have found this to be effective with extraction from different organisms

Binding

DNA binds to specific material

o Silica

o Chelex

Materials that bind DNA:

Silica

most common binding agent and the one found in the columns.

• has a property of binding or sticking to DNA.

prevent it from being washed out during the washing process.

Purpose of binding?

Acidic pH with high salt (Guanidinium)

DNA binding condition occurs in what pH?

Guanidinium

The solution loosens up the water quote surrounding the DNA so it denatures the DNA pole and helps shields the DNA's negative charges so that the DNA and the Silica can get close to each other and they will stick.

Isopropanol

Helps DNA and silica find each other & kick off the water

Washing

Removes other impurities to purify the DNA

o Buffer with ethanol

o Wash buffer

Wash solutions:

Buffer with ethanol

Buufer that dissolved salts, but not DNA, so you can wash the salt away

Crude method

DNA extraction that doesn't do any purification at all.

3 times

Washing is done how many times?

Elution

Release of DNA from the material to which it is bound

in order to transfer the DNA into the solution

Eluents:

o TE (Tris-EDTA) buffer

o Distilled water

Eluents:

Low salt, higher pH

Elution Conditions

Eluate

solution that comes out of elution.

• detergents

• phenol in high concentration

• Guanidine isothiocyanate

In RNA lysis what is used?

spectrophometer

We can quantify RNA by using

260 nm

What is the wavelength in quantifying DNA/RNA?

o DNA- 1 OD = 50ug/ml

o RNA- 1 OD = 40 ug/ml

Optical density used in quantifying of DNA and RNA

Nanodrop or qubit system

Other equipment we can use to quantify DNA

agarose gel electrophoresis

• best way to visualize the DNA that we have actually amplified or DNA that we just extracted.

True

True or false:

agarose gel that has two ends, 1 negative and the other is 1 positive.

False

True or false:

DNA are positive so they are attracted more to the positive ends of the electrode

On the negative side (black end)

When doing agarose gel electrophoresis, the sample is placed on what side?

those that travel farther from the well, they are smaller fragments and those that travel closer to the well, they are larger fragments

differentiate the different sizes of the fragments of DNA

2.5% agarose powder and dissolve it in 100ml of buffer

What is used to prepare an agarose?

TBE or TAE (Tris- Borate EDTA buffer or Tris-Acetic acid EDTA buffer)

What buffer is used in agarose

Gel loading buffer

What do we use to shoot the sample in the gel?

o 0.05% bromphenol blue

o 40% sucrose

o 0.1 M EDTA

o 0.5% SDS

Gel loading buffer -components:

DNA ladder (100bp)

Ladder we use to be able to estimate the sizes of the fragments that we can see.

• Ethidium bromide

• HiSyBR Green

Stain used in DNA