This experiment describes the procedure regarding radioactive autoradiography and the reading of DNA sequences from sequencing gels. This experiment comes from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Radiation autoradiography and DNA sequence reading experiments from sequencing gels Move makings For more product details, please visit Aladdin Scientific website.
Buffers and solutions
Formulations for storage solutions, buffers and reagents are given in Appendix 1.
Dilute the storage solution to the appropriate concentration.
Gel Fixative
300 ml Methanol
2.4L water
300 ml Glacial Acetic Acid
Radioactive mixture
Radioactive ink or chemiluminescent marker
See Appendix 9.
Reusable radioactive inks are primarily Stratagene's cold light markers (Glosos). The marker can be used multiple times and should be exposed to fluorescent light just prior to a new radiographic autoradiography cycle.
Gel
DNA Sequencing Gel
Preparation and electrophoresis are described in protocols 8 to 11.
Special equipment
Radiographic autoradiographic dark box (metal, spring 35.6 cmX43.2 cm)
Radiographic intensifier screen
Two large intensified calcium tungstate screens (e.g., LighteningPlus, Dupont) are required per film.
Gel dryer at 80°C
The gel dryer should be connected to a vacuum system.
Polyester film sheet (approx. 5 cm longer and wider than the gel)
The polyester film is used to ensure that the gel remains flat when drying under vacuum.
Saran Towel
Stainless steel metal spatula or scalpel
Tray for fixing the gel
The tray should be approximately 5 cm longer and wider than the gel.
Whatman3 MM CHR filter paper (or equivalent)
Whatman3 MM filter paper (or equivalent)
X-ray film (blue light sensitive 35 cmX43 cm)
For 32P-labeled DNA, use a double-layer film such as X-Omat-AR (Kodak) or BioMaxMS (Kodak). For 32P- and 35S-labeled DNA, use a single-layer film such as BioMaxMR (Kodak).
X-ray Film Processor
Methods
1. At the end of electrophoresis, turn off the power and disconnect the sequencing device from the power supply. Discard the power buffer and remove the film mold from the unit.
2. lay the gel mold flat on a protective lab cloth with plastic so that the smaller (slotted) plate is uppermost.
Allow the gel to cool to below 37°C before handling.
3. Remove any remaining gel sealing tape and use the end of a spatula to slowly and gently pry open the glass plate of the gel mold. The gel should be applied to the longer (non-slotted) glass plate.
WARNING: Wear safety glasses, as the glass plate can sometimes break during this step. Also, radioactive material may stick to the surface of the glass plate when it comes in contact with the gel and should be treated properly. 
Steps 4-13 can be omitted if the product of the sequencing reaction is labeled with 32P. For example, enzymatic sequencing is initiated with a 32P-labeled primer or [ a-32P ]dNTP is incorporated during extension of an unlabeled primer. When a pocket monitor is used to scan the gel surface, a reading of 20 to 100 cps indicates that there is enough radioactive material for staining. In this case, cover the gel with Saran wrap, making sure there are no wrinkles on it. Mark the orientation and expose the gel directly to the X-ray film. Leakage of radioactive liquid may occur during this procedure, so stay away from the gel and do not take the risk of saving a few hours of work.
The radioactivity of the labeled product in a Maxam-Gilbert sequencing reaction is usually insufficient for fixation and staining.
4. When the slide is separated, cut off the bottom or top corner of the sample-first side of the gel to orient the gel in subsequent operations.
5. fix the gel into a gel fixative (methanol/acetic acid; see Materials).
Sequencing gels containing 33P or 35S should be fixed before drying. This step is optional for gels labeled in 32P. Fixation improves resolution. It sometimes enhances the intensity of some of the radioactive signals and reduces excessive concentrations of urea or formamide from the gel. Fixation is also the last resort for removing the gel, since the gel is likely to stick to the two plates of the mold.
6. Transfer the gel (along with its supporting glass plate) to a shallow dish containing a methanol:acetic acid solution. Fix the gel at room temperature for 30 min. do not stir the liquid during fixation.
The thinner the gel, the faster it will fix and the thicker the gel, the slower it will fix. A 0.2 mm thick gel takes lOmin to fix, while a 0.6 mm thick gel takes 1b.
-A batch of fixative can be used to fix several gels. If the gel shows signs of falling off the supporting glass plate, cover it with a hard plastic mesh (available at many hardware stores) to prevent it from slipping or crumpling.
7. After 30 min, lift the glass plate very, very slowly from the fixative. Try to keep the glass plate as level as possible until most of the fixative has drained from the plate. Place the glass plate on a dozen sheets of paper with the gel side on top. Wipe any remaining fixative off the glass plate with Kimwipes. Be careful not to let the Kimwipes paper come in contact with the gel surface.
Then wear gloves to gently remove any wrinkles and tears in the gel.
8. Prepare a Whatman3 MMCHR filter paper (or equivalent) slightly larger than the gel in length and width (2-3 cm), keeping the paper in a bowed shape and placing the center of the bowed paper in contact with the center of the gel. Let the filter paper fall gently onto the surface of the gel, and then press gently so that the gel is firmly attached to the rough side of the filter paper.
9. Holding the filter paper in place with one hand, lift the supporting glass plate with the other hand and quickly turn it over, placing it on the lab bench glass plate up. Gently lift the glass plate upward to separate the Whatman3 MMCHR filter paper from the glass plate. When the glass plate is completely removed, the gel will be attached to the filter paper.
10. Place the Whatman3 MMCHK Filter Paper (gel side up) on top of two pieces of Whatman3 MM Filter Paper and cut a piece of Swan Wrap slightly larger and wider than the gel and place it on top of the gel. Try to avoid creases and air bubbles. This step is easier to accomplish with the help of another person. Hold the corners of the Sarari wrap and pull outward to stretch it taut. Place the flattened Saran wrap on top of the gel. Once the Saran wrap is in contact with the gel, do not attempt to lift it as this can tear the gel. The flat end of an agarose gel comb, Kimwipess paper, or a piece of plastic card can dislodge any air bubbles between the gel and the wrap.
This is chosen because placing a suitable piece of Saran wrap on the bench ensures that there are no wrinkles, and the gel can then be turned upside down so that the gel faces downwards towards the Saran wrap.
11. Using a paper cutter or a pair of scissors, trim all 3 sheets of Whatman filter paper and Saran wrap to the same size as the gel.
12. Place the sandwich consisting of the filter paper, gel and Saran wrap in the gel dryer. The packaging film is on top and a piece of mylar is used to keep the sandwich flat during drying.
13. Dry the gel under vacuum at 80°C for 30-60 min according to the dryer manufacturer's instructions.
Drying the gel shortens the distance at which the radioactive particles hit the X-ray film, thus increasing the sensitivity of the detection.
14. Remove the gel from the dryer and peel off the Saran wrap. The dried gel should be smooth to the touch and not sticky. If it is sticky, a remedy is to turn the latex glove over so that the inside of the talcum powder is facing outward, and use the inside of the glove to brush the gel surface with the talcum powder. To determine the orientation of the gel, a small sticky label labeled with radioactive or chemically fluorescent ink should be attached to the void created in the 3 MMCHR filter paper after the bottom corner of the gel has been cut off (Step 4).
15. In a darkroom, place the dried gel in a spring-loaded metal box. Cover with a piece of unexposed X-ray film and close the dark box. Perform radiographic autoradiography for 16 to 24 h at room temperature or -80°C.
The optimal exposure time required for a piece of sequencing film is difficult to predict precisely. 16-24 h may be a bit long if every piece of RC is doing well during the sequencing reaction due to 5 or more factors. In less favorable situations, longer exposure times are usually required. The main purpose at this point is to establish one or more off-chip exposures depending on whether the sequencing reaction is going well and to look at the intensity of the bands.
16. Develop the radiographic autofilm according to the film manufacturer's requirements and read the DNA sequence as described below. 