TITLE: 2 Methods for DNA Adsorption on a Mica Substrate for AFM Imaging in Fluid
AUTHORS: Philip J. Haynes, Kavit H. S. Main, Alice Pyne

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is part 2 of the "Atomic Force Microscopy of DNA and DNA-Protein Interactions" collection of protocols.</div><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract:</span><span> Atomic force microscopy (AFM) is a microscopy technique that uses a sharp probe to trace a sample surface at nanometre resolution. For biological applications, one of its key advantages is its ability to visualize substructure of single molecules and molecular complexes in an aqueous environment. Here, we describe the application of AFM to determine the secondary and tertiary structure of surface-bound DNA, and it’s interactions with proteins.</span></div></div>

[STEPS]
?. The three Methods for DNA Adsorption on a Mica Substrate for AFM Imaging in Fluid are outlined below:
?. [2.1 DNA Adsorption Using Divalent Cations]
Divalent cations (in this case Ni2+) can be used to overcome the electrostatic repulsion between DNA and mica, thus facilitating DNA adhesion to the mica, which can also be tuned via the cationic concentration in the solution as outlined below.
?. [2.1 DNA Adsorption Using Divalent Cations]
Cover the freshly cleaved mica with  (see Note 10).
[nickel adsorption buffer]
?. [2.1 DNA Adsorption Using Divalent Cations]
Add  (, see Note 11) and distribute evenly in the meniscus by gently purging.
[DNA]
?. [2.1 DNA Adsorption Using Divalent Cations]
Adsorb for . Then gently exchange the buffer to the nickel imaging buffer four times to remove any unbound DNA.
?. [2.1 DNA Adsorption Using Divalent Cations]
Add sufficient nickel imaging buffer to form a droplet covering the sample (dependent on the AFM system, see Note 12).
?. [2.1 DNA Adsorption Using Divalent Cations]
Mount sample on AFM.
?. [2.1 DNA Adsorption Using Divalent Cations]
Gently exchange the buffer to the nickel imaging buffer to remove any unbound DNA. (1/4)
?. [2.1 DNA Adsorption Using Divalent Cations]
Gently exchange the buffer to the nickel imaging buffer to remove any unbound DNA. (2/4)
?. [2.1 DNA Adsorption Using Divalent Cations]
Gently exchange the buffer to the nickel imaging buffer to remove any unbound DNA. (3/4)
?. [2.1 DNA Adsorption Using Divalent Cations]
Gently exchange the buffer to the nickel imaging buffer to remove any unbound DNA. (4/4)
?. [2.1 DNA Adsorption Using Divalent Cations]
Immediately before DNA adsorption, cleave a 6 mm mica disc that has been prepared as described in protocol 1.
?. [2. DNA Adsorption Using PLL]
DNA adsorption on mica can also be assisted by surface modification of mica. Poly- L-lysine creates a cationic monolayer on the mica surface due to its protonated amino groups. DNA can then bind to the positively charged groups. The procedure for this is outlined below.
?. [2. DNA Adsorption Using PLL]
Immediately before exposure to poly-L-lysine, cleave a mica disc that has been prepared as described in protocol 1.
?. [2. DNA Adsorption Using PLL]
Cover the mica with  (, 150,000-300,000 MW).
[poly-L-lysine solution]
?. [2. DNA Adsorption Using PLL]
Incubate for .
?. [2. DNA Adsorption Using PLL]
Hold the disc at an angle and thoroughly rinse under a stream of ultrapure water. Or, equivalently, wash 5x with ultrapure water.
?. [2. DNA Adsorption Using PLL]
Blot the disc and add .
[poly-L-lysine imaging buffer]
?. [2. DNA Adsorption Using PLL]
Add  (, see Note 11) and distribute evenly in the meniscus by gently purging.
[DNA]
?. [2. DNA Adsorption Using PLL]
Absorb for  (see Note 3). Then wash four times with the poly-L-lysine imaging buffer to remove any unbound DNA (see Note 13).
?. [2. DNA Adsorption Using PLL]
Wash with the poly-L-lysine imaging buffer to remove any unbound DNA. (1/4)
?. [2. DNA Adsorption Using PLL]
Wash with the poly-L-lysine imaging buffer to remove any unbound DNA. (2/4)
?. [2. DNA Adsorption Using PLL]
Wash with the poly-L-lysine imaging buffer to remove any unbound DNA. (3/4)
?. [2. DNA Adsorption Using PLL]
Wash with the poly-L-lysine imaging buffer to remove any unbound DNA. (4/4)
?. [2. DNA Adsorption Using PLL]
Add sufficient poly-L-lysine imaging buffer on the sample (dependent on the AFM system, see Note 12).
?. [2. DNA Adsorption Using PLL]
Mount sample on AFM.
?. [3. DNA Adsorption Using PLL-b-PEG]
The poly(L-lysine)-b-poly(ethylene glycol) (PLL-b-PEG) block copolymer, PLL10-b-PEG113 (subscript refers to the number of monomer repeats), can be purchased as a lyophilized powder and dissolved in ultrapure water to 1 mg/mL on the day of imaging (see Note 14). It is supplemented 1:1 with poly-L-lysine solution (PLL1000-2000, 0.01% w/v, 150,000-300,000 MW) to aid DNA adsorption. The significant decrease in non-specific background protein with the addition of PLL-b-PEG is shown in Fig. 5b and 5c. The substrate preparation is outlined below.
?. [3. DNA Adsorption Using PLL-b-PEG]
Prepare a ≥ 1:1 PLL10-b-PEG113/PLL1000-2000 (PLL-b-PEG/PLL) mixture in an Eppendorf.
20 µl
?. [3. DNA Adsorption Using PLL-b-PEG]
Cleave a mica disc that has been prepared as described in protocol 1.
?. [3. DNA Adsorption Using PLL-b-PEG]
Immediately after cleaving, add  and leave to incubate in a humidified petri dish for  (see Note 15).
[PLL-b-PEG/PLL mixture]
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash 5x with ultrapure water and a further 5 times with poly-L-lysine imaging buffer (see Note 3).
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with ultrapure water. (1/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with ultrapure water. (2/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with ultrapure water. (3/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with ultrapure water. (4/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with ultrapure water. (5/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with poly-L-lysine imaging buffer. (1/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with poly-L-lysine imaging buffer. (2/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with poly-L-lysine imaging buffer. (3/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with poly-L-lysine imaging buffer. (4/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with poly-L-lysine imaging buffer. (5/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Add  (, , see Notes 11 and 16), with or without protein, to the disc and gently mix. If adsorbing DNA with protein, the DNA and protein must be pre-incubated at /ambient temperature before deposition.
[496 bp linear DNA]
0 Room temperature
?. [3. DNA Adsorption Using PLL-b-PEG]
Incubate the sample on the disc for  and, with the imaging buffer, wash 5x and make up to  (see Note 12).
30 µl
?. [3. DNA Adsorption Using PLL-b-PEG]
Incubate the sample on the disc for .
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with the imaging buffer. (1/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with the imaging buffer. (2/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with the imaging buffer. (3/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with the imaging buffer. (4/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Wash with the imaging buffer. (5/5)
?. [3. DNA Adsorption Using PLL-b-PEG]
Make up to .
30 µl
?. [3. DNA Adsorption Using PLL-b-PEG]
Mount sample on AFM, approach and image as described in protocol 3.
?. [3. DNA Adsorption Using PLL-b-PEG]
For protein exchange assays where you add protein to a surface with DNA already adsorbed, carry out a buffer exchange for protein in the imaging buffer (see Note 17). Re-approach and resume imaging after a 5 minute incubation.