Bio-Rad Zeta-Probe Membranes User Manual

Zeta-Probe

Blotting Membranes

Instruction Manual

Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547

LIT234 Rev C

Table of Contents

Section 1 Introduction ..........................................................1

Section 2 Nucleic Acid Blotting Protocols ..............................1

2.1 Southern Blotting (DNA Capillary Transfer) .............2

2.2 Northern Blotting (RNA Capillary Transfer)..............3

2.3 Alkaline Blotting (DNA Capillary Transfer)................4

2.4 Electrophoretic Transfer .........................................5

2.5 DNA Dot Blotting ...................................................8

2.6 RNA Dot/Slot Blotting ............................................9

2.7 DNA Alkaline Fixation ...........................................10

Section 3 Probe Recommendations ....................................10

Section 4 Hybridization Protocols for DNA Probes...............11

4.1 Standard Protocol................................................13

4.2 Formamide Protocol ............................................15

4.3 Alternative Protocol..............................................16

4.4 Oligonucleotide Protocol ......................................17

Section 5 Hybridization Protocols for RNA Probes...............19

Section 6 Probe Stripping and Rehybridization ....................21

Section 7 Troubleshooting...................................................22

7.1 Nucleic Acids.......................................................22

Section 8 Appendix .............................................................26

Section 9 References ..........................................................28

Section 10 Ordering Information ............................................29

Section 1 Introduction

Zeta-Probe blotting membranes are nylon membranes which have unique binding and handling properties that make them ideally suited for nucleic acid, and some protein, blotting applications.

Zeta-Probe membranes possess a high tensile strength. They won't shrink, tear, or become brittle during transfer, baking, hybridization, or reprobing. Zeta-Probe membranes are heat-resistant, nonflammable, and autoclavable. Zeta-Probe membranes are naturally hydrophilic with no added wetting agents. These membranes are resistant to a wide variety of chemicals, including 100% formamide, 2 M NaOH, 4 M HCl, acetone, most alcohols, DMSO, DMF, and chlorinated aliphatic hydrocarbons. The nominal porosity of Zeta-Probe membranes is

0.45 µm. When stored at 23–25°C, Zeta-Probe membranes are stable for at least 1 year.

When handling Zeta-Probe membranes, always wear gloves or use forceps. After blotting, do not allow wet membranes to come in contact with each other. Contact may result in the transfer of blotted nucleic acids or proteins from one membrane to the other.

Stock buffers are listed in the appendix. It is suggested that you read the entire protocol before proceeding.

Section 2 Nucleic Acid Blotting Protocols

Several nucleic acid blotting methods are presented in this section. Capillary blotting (Sections 2.1through 2.3) is generally used with agarose gels, and electrophoretic transfer (Section 2.4) is used with polyacrylamide gels. Dot blotting (Sections 2.5 and 2.6) is used for

diagonally and aligning the opposite corners with the gel corners. Then lower the Zeta-Probe membrane onto the gel.

6. Cut two pieces of 3MM paper to the size of the gel. Place both sheets on top of gel. Wet paper with small amount of transfer buffer. Roll with the pipet to remove air bubbles.

7. Flood the surface of the gel with buffer. Carefully place paper towels over the Whatman paper. Stack the paper towels about 15 cm high.

8. Cover the paper towel stack with a glass or plastic plate. Keep the pressure on the paper towel stack at a minimum. Excessive weight will compress the gel, retarding capillary transfer.

9. Keep an excess of buffer in the dish, but do not cover the top of the sponge. Continue transferring for 2–24 hr, depending on the gel concentration and fragment size.

10. After transfer, separate the membrane from the gel, rinse the membrane briefly in 2x SSC, and briefly blot the membrane with filter paper. The DNA can then be fixed onto the Zeta-Probe membrane by baking it at 80°C for 30 min in a vacuum oven. Alternatively, the DNA can be UV-crosslinked to the membrane using 5,000 µJ/cm

radiation. Higher levels, although they increase the absolute retention of the nucleic acid on the membrane, can lead to a reduction in signal intensity. The membranes can be stored dry between two pieces of filter paper in plastic bags at 23–25°C.

2.2 Northern Blotting (RNA Capillary Transfer)

Follow the Southern blotting protocol (Section 2.1), omitting steps 1–3. No pretreatment of RNA gels is necessary.

If gels contain glyoxal, remove glyoxal adducts by vacuum baking Zeta-Probe membrane for 1 hour at 80°C after transfer. Alternatively,

nucleic acids in solution. DNA alkaline blotting (Section 2.3) is an alternative to Southern blotting. DNA alkaline blotting results in higher resolution and greater sensitivity in many applications. DNA alkaline fixation (Section 2.7) can be used to denature and covalently fix DNA to Zeta-Probe membranes after transfer.

2.1 Southern Blotting

1,2

(DNA Capillary Transfer)

1. Depurinate the DNA by soaking the gel in 0.25 M HCl for 10–15 min (be sure that the gel is floating free in all baths).

Note: Acid depurination is only recommended for fragments >4 kb.

2. Denature the DNA by placing the gel in a bath of 0.5 N NaOH, 1 M NaCl. Place the container on a moving platform for 30 min at room temperature.

3. Neutralize the gel by bathing it in 1.5 M Tris-HCl, pH 7.4,

1.5 M NaCl for 30 min at room temperature on a moving platform. Prepare a Whatman 3MM paper wick. Hang two sheets, prewetted with 10x SSC hung over the sides and into the bottom of the capillary transfer apparatus containing 800 ml 10x SSC. On top of the wick, place two additional sheets of 3MM paper cut to the size of the gel prewetted with 10x SSC.

4. Invert the gel and place it on the wick. Roll a 10 ml plastic pipet, over the gel to remove any bubbles. Trim off the wells from the gel using a spatula.

5. Place membranes labeled side against the gel above the lanes to be transferred. Trim edges of the gel as required with a spatula, or cover exposed areas with Parafilm. Roll with the pipet to remove any air bubbles. It is important to remove air bubbles from underneath the blotting membrane as they will block transfer. To avoid trapping bubbles, place the Zeta-Probe membrane onto the gel surface by first bowing the membrane

pour 95°C 20 mM Tris-HCl, pH 8.0, 1 mM EDTA onto the blotted membrane, then gently agitate at room temperature until the solution cools. After removal of glyoxal adducts, proceed to hybridization or store the membranes dry.

2.3 Alkaline Blotting3(DNA Capillary Transfer)

1. Depurinate the DNA by soaking the gel in 0.25 M HCl for 10–15 min. Rinse the gel several times with distilled water.

Note: Acid depurination is only recommended for fragments >4 kb.

2. Cut four sheets of Whatman 3MM paper so they overhang the bottom of the gel tray by 5 cm on each end. Prewet the ZetaProbe membrane in distilled water.

3. Place the four sheets of 3MM paper on an inverted gel casting tray. Place the 3MM/tray in the bottom of a deep dish. Then saturate the 3MM paper with 0.4 M NaOH. Remove the bubbles by repeatedly rolling a glass pipet over the saturated 3MM paper. Pour enough NaOH into the deep dish so that the 3MM wick ends are immersed in NaOH.

4. Pour more NaOH onto the 3MM wick to saturate it, then carefully place the gel on the wick. Make sure that no bubbles are trapped beneath the gel. Cover the gel with a small amount of NaOH.

5. Place plastic wrap (such as Saran wrap) over the entire gel/3MM stack. Cut out a window with a clean razor blade, allowing only the gel to be exposed.

6. Lower the sheet of pre-wetted Zeta-Probe membrane onto the gel surface, making contact first in the center, then allowing the edges to gradually fold down. Carefully flood the filter surface with NaOH. Make sure that no bubbles are present between the gel and the Zeta-Probe membrane.

7. Cut two pieces of 3MM exactly to the gel size. Wet a sheet of precut 3MM paper in water and place it on the Zeta-Probe membrane/gel stack, then repeat with the second sheet. Remove any bubbles from beneath each layer of 3MM paper.

8. Place a stack of precut paper towels on the 3MM/Zeta-Probe membrane/gel stack. Cover the paper towel stack with a plastic or glass plate. Keep the pressure on the paper towel stack at a minimum. Excessive weight will compress the gel, retarding capillary transfer.

9. Continue transferring for 2–24 hours, depending on the gel concentration and fragment size. Note: Higher background may appear if transfer is longer than 24 hr.

10. After transfer, remove the stack of paper towels. Gently peel the Zeta-Probe membrane from the surface of the gel, rinse it in 2x SSC, and air dry. DNA is fixed to the membrane during transfer, eliminating the need for subsequent fixation. The dried membranes are stable at room temperature. The membranes can be stored dry between two pieces of filter paper in plastic bags at 23–25°C.

2.4 Electrophoretic Transfer

The following protocol was developed for maximum efficiency of electrophoretic transfer. It affords the greatest mobility of DNA and RNA, and the most complete transfer from gel to membrane without excessive heat generation. The buffer (ionic strength and pH) and field strength have been optimized for electrophoretic blotting of DNA and RNA from both agarose and acrylamide gels. For electrophoretic transfer from agarose gels, a heat exchanger must be used, because increased temperatures could melt the agarose gel. The protocol was developed using the Trans-Blot heat exchanger.

electrophoretic transfer system with a

Soak one fiber pad by squeezing it while it is submerged in 0.5x transfer buffer. Lay the soaked pad on the open gel holder. Soak a piece of thick filter paper (e.g., slab gel dryer type paper cut to the size of the fiber pad) in the transfer buffer and place it on the fiber pad. Place the gel on the filter paper. Hold the presoaked Zeta-Probe membrane with both hands so that the middle of the membrane is sagging or bowed downward. Allow the middle of the membrane to contact the gel first. Gradually lower the ends of the membrane onto the gel. This process will expel most bubbles from between the gel and the membrane. If there are any remaining bubbles between the gel and membrane, remove them by sliding a test tube or extended gloved finger across the surface.

Note: Maintaining uniform physical contact between the gel and membrane is of critical importance in electrophoretic transfer.

Place a presoaked piece of thick filter paper on the membrane followed by a presoaked fiber pad. Close the gel holder and place it in the transfer cell so that the membrane is on the anode side of the gel (red pole). Add more 0.5x transfer buffer, if necessary, to bring the buffer level to 1 cm below the electrode post.

6. Transfer at 80 V for 4 hours. Note: For comprehensive electrophoretic transfer

instructions, including protocols, technical discussion, and troubleshooting guide, refer to the Trans-Blot cell operating manual.

7. After transfer, separate the membrane from the gel, rinse the membrane briefly in 1x transfer buffer, and briefly blot

1. Prepare the stock electrophoretic transfer buffer, 20x TAE or 5x TBE.

2. Prepare gels for transfer immediately after electrophoresis: A. Electrophoresis Under Denaturing Conditions

If gel electrophoresis was done under denaturing conditions (e.g., agarose/formaldehyde gels), equilibrate the gel in 0.5x transfer buffer for 10–15 min prior to electrophoretic transfer.

B. Electrophoresis Under Nondenaturing Conditions

1. Soak the gel in 0.2 N NaOH, 0.5 M NaCl for 30 min. For polyacrylamide gels, be sure not to exceed 30 min, since limited gel hydrolysis may occur with subsequent swelling during transfer.

Note: Zeta-Probe membranes will bind nondenatured nucleic acids. Therefore, denaturing is not mandatory before transferring. Yet, after transferring, the blotted Zeta-Probe membrane must be treated with NaOH. Refer to the DNA alkaline fixation procedure (Section

2.7).

2. After base treatment, neutralize the gel by washing in 5x transfer buffer two times, 10 min each. Then wash the gel once in 0.5x transfer buffer for 10 min.

3. While gels are being equilibrated, soak the Zeta-Probe membrane at least 10 min in 0.5x transfer buffer.

4. Fill the electrophoretic transfer cell to half full with 0.5x transfer buffer, and circulate 4°C coolant through the heat exchanger. If possible, place the cell on a magnetic stirring plate and add a stirbar. Circulate buffer in the cell by stirring to maintain uniform temperature during the run.

5. Prepare the transfer assembly.

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