Polyacrylamide gel electrophoresis (PAGE) experiment.

study and master
working principle and operating techniques of polyacrylamide gel electrophoresis in the course of the experiment.
:
Polyacrylamide gel electrophoresis (polyacryamide gel electropHoresis, PAGE) is a three-dimensional mesh structure substance formed by acrylamide monomer and crosslinker methicilline in catalysis. In the electrophoresis of non-continuous polyacrylamide gels, the production of gels is layered, so the gel not only has a molecular sieve effect, but also has a concentration effect.
polyacrylamide gel
s
difficult to prepare and treat compared to agarose gels, polyacrylamide gels are difficult to prepare and treat. Their separation range is narrow. However, they also have outstanding advantages, because of the non-continuous pH gradient, so the sample is compressed into a narrow zone, thus enhancing the separation effect, improve electrophoresis resolution. In particular, the analysis
fragments
small DNA fragments (5 to 500bp).
this range, only 1bp of DNA molecules can be clearly separated. Second, DNA is very pure. The DNA from the polyacrylamide gel is so pure that no treatment is required for the next step. Also, it has a high load capacity. Up to 10mL of DNA samples can be added to the glue's standard sample slot without affecting electrophoresis resolution.
used to isolate purified and identify DNA fragments with a size of 5 to 500bp. Polyacrylamide gel electrophoresis has two kinds of continuous and seconcum system, the former refers to the buffer pH and gel aperture size in the whole electrophoresis system is the same, mainly used for nucleic acid analysis.
The latter is mainly used for the separation of
protein
samples, and in addition to the buffer system and pH in the electrophoresis tank, the gel itself is also composed of two gels with different buffer systems, pH values and gel apertures. The following main introduction to the continuous system.
instruments, materials and
reagents1.30% acrylamide/N, N'-methyl diacrylamide (Acr/Bis): 29g acrylamide, 1g N, N'-methyl diacrylamide, distilled water to 100mL.
2.10% ammonium persulphate (Aps): 0.4g ammonium persulphate, 4mL distilled water, TEMED (N, N, N', N'-tetramethyl ethyl diamine).
3.1.5mol/L Tris-HCI (pH8.8): Take 3mol/L Tris master liquid 50mL, adjust pH to 8.8 with hydrochloric acid, and set the capacity to 100mL (4 degrees C) with heavy steamed water Storage); 0.5mol/L Tris-HCI (pH6.8): Take 1mol/L Tris solution 50mL, adjust pH to 6.8 with hydrochloric acid, and hold to 100mL (4 oC storage) with re steamed water.
4.10% sodium tetranyl sulfate (
SDS
) (stored at room temperature).
5.1×TBE buffer: 89mol/LTris-borate, 2mol/L EDTA (pH8.0).
6.I sample buffer: 0.25% bromophenol blue, 0.25% xylene green FF, 40% sucrose aqueous solution (W/V) 4 degrees C storage. Back cover glue: 1% agar solution (configured with distilled water).
7. Staining liquid: 0.2g Kaomas bright blue R-250, 84mL95% ethanol, 20mL ice acetic acid, fixed capacity to 200mL,
filter
spare.
8. Decoloring liquid: Medical alcohol: ice acetic acid: water: 4.5:0.5:5 (V:V:V).
9. Preserving liquid: 7% ice acetic acid.
10. Glass plate, partition, electrophoresis tank, beech, magnetic mixer, microwave oven or
heating
,
balance
, comb, UV analyzer, power supply, etc.
:
continuous system
preparation of 100mL gel liquid

distilled water 37.5mL
microwave oven heating 30s, mixing stick stirring
urea 50g
10×TBE buffer 8mL×
30% acrylamide 10mL
before pouring glue
10% ammonium persulphate 0.8mL
TEMED 23.5 μL
2. Clean a short two glass plates with detergent and insert a space sheet between the two plates, then dry with ethanol.
3. Align the length of the two plates on one side, insert a spacer approximately 1.5cm wide, and clamp the glass plates on all three sides with a clip. Seal the three sides of the plywood with a back cover glue (1% agar solution).
4. Add 10% ammonium persulphate and TEMED to mix in the glue solution and fill immediately. To avoid bubbles, be careful and slow when pouring glue. If bubbles are produced, tap the plywood from bottom to top with a rubber hammer or pencil head to remove the bubbles.
5. When added to a short glass plate margin of about 0.5cm, stop the adhesive. Gently insert a comb of the right size, do not allow bubbles to bubble under the comb, clamp the comb and plywood, and then add the remaining glue to both sides of the comb.
6. Room temperature condensation time is about 0.5 to 1h.
7. Carefully remove the comb, wash the sample hole with water, secure the glue to the electrophoresis tank, add 1 × TBE buffer.
8. Mix DNA samples with 6x I-type sample buffers. Add 3 to 5/L of the mixture per hole. The voltage gradient for electrophoresis is 1V/cm to 8V/cm.
9. After electrophoresis, remove the glue and place it in the EB to dye 30min. The EB concentration is (0.5 μg/mL 1x TBE buffer).
10. Analysis and photography in ultraviolet light.
(the seism system is mainly used for the analysis of proteins, the process can be seen in Chapter III, so here is not introduced.) (
)
1. Preparation gels should be made with high purity reagents, otherwise the effect of gel solidification and electrophoresis should be affected. Both Acr and Bis are nerve agents that irritate the skin, wear gloves and masks when operating, and purification should be carried out in the ventilation cabinet.
. Do not touch the glass of the gel surface by hand to prevent the gel plate and glass plate from peeling off, creating bubbles and slippage, or the gel plate can break easily when peeling the glue. Therefore, the equipment used should be strictly cleaned.
3. There should be no bubbles when using agarose back cover and gel, so as not to affect the current flow during electrophoresis.
. In order to prevent the tailing of the electrophoresis rear area, the salt ion strength in the sample should be reduced as much as possible. Samples with high salt content can be desalinated by dialysis or gel filtration.
5. After the gel is fully solidified, it must be placed about 30min, so that it is fully "aging", before gently removing the sample comb, do not destroy the flatness of the bottom of the sample hole, so as not to distort the area after electrophoresis..