Agarose Gel Electrophoresis Protocol
( Project1: PCR Product)
1. Materials and Equipment
TBE buffer (receipt see below)
Agarose power
SH2O
Microwave
Flask
gel box.
Comb
Gel tray
2. Preparation
TBE buffer?
3. Procedure (project1)
3.1 Receive the sheet
3.2 Preparing the agarose gel (0.8%):
1) For a small gel, measure 150 mL of 1X TBE buffer in a graduated cylinder and pour it into a 500 mL flask.
2) Weigh 1.2 grams of electrophoresis grade agarose and add it to the buffer solution in the flask and swirl it gently.
NOTE: 150ml gel solution can get: 4 of 24 well solid gel
3) No keeping the cap of flask too tight .
3) Adjust the level of fire to mediate-high and place the flask into a microwave..and turn off the door.
.5) Heat 4 min, take out the flask by glove.
4) Cool the agarose to about 50-55°C in a water bath for about 5-10 minutes (until it doesn’t burn the surface of your inner forearm).
5) Swirl the agarose occasionally while it cools.
6) Place the gel tray into the gel box so that the ends of the tray are blocked by the sides of the box
perpendicular to the ends of the box.
7) Place the comb in the slots on one end of the tray.
8) Pour the cooled agarose into the tray and there is an equal amount of buffer solution in either chamber .The thickness is about 3mm.
9) Let it cool until it is solid (it should appear milky white).
10) Carefully pull upward on the comb to remove it once the agarose has solidified.
11) Remove the tray from the box into the electrophoresis box.the rest of gel is stored in a membrane.
11) Pour buffer solution into the electrophoresis box and so there is about 2-3 mm of solution above the gel.
3.3 Loading the gel:
Project Link Sheet(2-3)
Project ID:
Date:
From: PCR
To : Electrophoresis
Task:
Electrophoresis of PCR products
Number of PCR products tubes
1 2 3 4
Size of every PCR product
| 1st | 2nd | 3rd | 4th | |
| identity | ||||
| size |
M1:marker name, 5ul
1:identity 1 , 5ul
2:identity 2 , 5ul
3:identity 3 , 5ul
4:identity 4 , 5ul
M2:marker name, 5ul
photo
M1 1 2 3 4 M2
electrophoresis result:
Annotation:
OK NG
1) Place 1μl of loading dye onto a square of Parafilm.
2) Pipette 5ul PCR reaction onto this 1μl spot of loading dye and add the 1xTBE up to 10ul.
Sample : 5ul + 1X TBE :5ul + loading dye: 1ul
3) Slowly pipette the mixture up and down to mix it.
3) Reset your micropipette to 10 μl and carefully pipette the PCR/loading dye mixture into a well
of your gel.
4) Pipette 10 μl of DNA ladder into another well of the gel.
5) Draw a map in your laboratory notebook with labels to indicate which well contains your PCR
products as well as the DNA ladder.
3.4 Running the gel:
1) Connect the lid to the gel box.
2) Make sure that the negative pole is on the same side as the wells of your gel.and then connect the positive and negative leads into the appropriate connection on the power supply.
2) Turn the power supply on and adjust the voltage to about 120 volts.
3) Check that the loading dye is running toward the correct end of your gel after a few minutes.
4) Run the gel until the first dye is near the end of your gel.
5) Switch the power supply off, remove the lid, and carefully lift your gel tray out.
Staining and photographing the gel:
6) Carefully place your gel into the staining tray with the gel stain (ethidium bromide).
7) Let the gels stain for 10-20 minutes.
8) Carefully remove the gel. place it in a glass dish and rinse it 2-3 times with distilled water.
9) Use a spatula to place the gel on the UV light source and photograph it.
10 Dispose of the gel in the biohazard waste container when finished.
11) Fill in the sheet and load the picture to the database
12) Send the sheet to the another department.
Agarose Gel Electrophoresis Protocol
(Project2: Enzyme Digestion)
1.Materials and Equipment ( see project1)
2.Preparation( see project1)
3.Procedure
3.1Receive the following sheet
Project Link Sheet(2,4-3-5)
Project ID:
Date:
From: PCR/Vector
To : Electrophoresis
Task:
PCR clean up gel extracton of linearized vector
Elution volume:
30ul 40ul 50ul
photo
electrophoresis result:
M:DL2,000, 5ul
1: vector , 2ul
2: PCR product , 2ul
3-8:diluted plasmid series,
1 2 M 3 4 5 6 7 8
Quantification:
Linearized vector: ( )ng/ul
PCR product: ( )ng/ul
From:electrophoresis
To :clone
Task:
Infusion ligation
3.2 Preparing the agarose gel (See Project1)
3.3 Loading the gel
2) Place 1μl of loading dye onto a square of Parafilm.
3) Pipette 5ul enzyme digested reaction onto this 1μl spot of loading dye and add the 1xTBE up to 10ul.
Sample: 5ul + 1XTBE: 5ul + loading dye: 1ul
3) Slowly pipette the mixture up and down to mix it.
3) Reset your micropipette to 10 μl and carefully pipette the PCR/loading dye mixture into a well
of your gel.
4) Pipette 10 μl of DNA ladder into another well of the gel.
5) Draw a map in your laboratory notebook with labels to indicate which well contains your PCR
products as well as the DNA ladder.
3.4 Running the gel( see project1)
Agarose Gel Electrophoresis Protocol
(Project3: Quantity Identify )
1.Materials and Equipment ( see project1)
2.Preparation( see project1)
3.Procedure
3.1Receive the following sheet
Project Link Sheet(2,4-3-5)
Project ID:
Date:
From: PCR/Vector
To : Electrophoresis
Task:
PCR clean up gel extracton of linearized vector
Elution volume:
30ul 40ul 50ul
photo
electrophoresis result:
Quantification:
Linearized vector: ( )ng/ul
PCR product: ( )ng/ul
M:DL2,000, 5ul
1: vector , 2ul
2: PCR product , 2ul
3-8:diluted plasmid series,
1 2 M 3 4 5 6 7 8
From:electrophoresis
To :clone
Task:
Infusion ligation
3.2 Preparing the agarose gel (See Project1)
3.3 Loading the gel
1) Fill the sheet and prepare to load the sample.
2) Place 1μl of loading dye onto a square of Parafilm.
3) Pipette 2ul vector and PCR product onto this 1μl spot of loading dye and add the 1xTBE up to 10ul.
Sample:2 ul + 1XTBE: 8ul + loading dye: 1ul
3.4 Running the gel ( see project1)
Agarose Gel Electrophoresis Protocol
(Project4:PCR Product )
1 Materials and Equipment ( see project1)
2. Preparation( see project1)
4.Procedure
3.1 Receive the following sheet
3.2 Preparing the agarose gel (See Project1)
3.3 Loading the gel
2) Place 1μl of loading dye onto a square of Parafilm.
3) Pipette 2ul vector and PCR product onto this 1μl spot of loading dye and add the 1xTBE up to 10ul.
Sample: all sample :25ul + 2ul loading dye 10ul of mixture to loaded
Or
| What is Electrophoresis? | |
Electrophoresis is a technique used in the laboratory that results in the separation of charged molecules. DNA is a negatively charged molecule, and is moved by electric current through a matrix of agarose. | |
| What is a Gel? | |
Purified agarose is in powdered form, and is insoluble in water (or buffer) at room temperature. But it dissolves in boiling water. When it starts to cool, it undergoes what is known as polymerization. Rather than staying dissolved in the water or coming out of solution, the sugar polymers crosslink with each other, causing the solution to "gel" into a semi-solid matrix much like "Jello" only more firm. The more agarose is dissolved in the boiling water, the firmer the gel will be. While the solution is still hot, we pour it into a mold called a "casting tray" so it will assume the shape we want as it polymerizes (otherwise it will just solidify in the bottom of the flask wasting the expensive agarose). | |
| DNA Electrophoresis Agarose gel electrophoresis can be used to separate DNA molecules according to size. In fact, we load a dye with our DNA samples so that we can monitor the progress of the electrophoresis. The dye solution also contains glycerol to provide a dense mixture that will stay in the bottom of the well. Materials DNA samples DNA size standard Loading dye solution Agarose TBE buffer Sybr Green I solution diluted 10,000-fold Dark Reader light box Plastic wrap | |
| Procedure | |
| :: | Pour an agarose gel and cover it with TBE buffer. |
| :: | Pipette 5 ul drops of the loading dye solution onto a piece of plastic wrap (one for each DNA sample) |
| :: | Pipette 5 ul of each DNA sample into one of the drops of loading dye. Mix by pipetting up and down and carefully load into a well of the agarose gel. |
| :: | Pipette 10 ul of a solution containing a DNA size standard into an empty well of the gel. |
| :: | Place the lid on the gel box and turn on the power supply to 100 volts. |
| :: | After the tracking dye has migrated half to two thirds of the way through the gel, turn off the power, remove the gel from the gel box and place in a tray containing enough Sybr Green I solution to cover the gel. Swirl occasionally. After 15 minutes, remove the gel from the staining solution and place on the light box. Observe the gel through the orange filter in a dimly lit room. |
| :: | Save the Sybr Green I solution. It can be reused several times. |
| 10 Fun Facts of DNA Electrophoresis | |
| :: | When preparing agarose for electrophoresis, it is best to sprinkle the agarose into room-temperature buffer, swirl, and let sit at least 1 min before microwaving. This allows the agarose to hydrate first, which minimizes foaming during heating. |
| :: | Electrophoresis buffer can affect the resolution of DNA. TAE (Tris-Acetate-EDTA) buffer provides better resolution of fragments >4 kb, while TBE (Tris-Borate-EDTA) buffer provides better resolution of 0.1- to 3-kb fragments. In addition, use TBE buffer when electrophoresing >150 V and use TAE buffer with supercoiled DNA for best results. |
| :: | Migration of DNA is retarded and band distortion can occur when too much buffer covers the gel. The slower migration results from a reduced voltage gradient across the gel.. |
| :: | Loading DNA in the smallest volume possible will result in sharper bands. |
| :: | You can preserve DNA in agarose gels for long-term storage using 70% ethanol. [See Jacobs, D. and Neilan, B.A. (1995) BioTechniques 19, 2.] |
| :: | Electrophoresing a gel too "hot" can cause the DNA to denature in the gel. It can also cause the agarose gel to deform. Cool the gel with a small fan during the electrophoresis. |
| :: | For the Supercoiled DNA Ladder electrophoresed on <1% agarose gels, add 2 1lg/ml ethidium bromide to the gel. Otherwise, smeared bands and extra bands will be seen because of different degrees of supercoiling. [See Longo, M.C. and Hartley, J.L. (1986) ] |
| :: | When glycerol-containing loading buffers are used in DNA samples electrophoresed through acrylamide gels, smiling bands may be accentuated especially in TBE. |
| :: | On a polyacrylamide gel, DNA fragments having AT-rich regions migrate slower than other DNA fragments of the same size. This anomalous migration is enhanced at lower temperatures and disappears at high temperatures. This anomalous migration is not observed on agarose gels. [See Stellwagen, N.C. (1983) Biochemistry22, 6186.] |
| :: | The minimum amount of DNA detectable by ethidium bromide on a 3-mm-thick gel and a 5-mm-wide lane is 1 ng. Do not exceed 50 ng of DNA per band on a 3-mm-thick gel and 5-mm-wide lane. |
| Troubleshooting DNA Agarose Gel Electrophoresis | |
If you see faint or no bands on the gel: | |
| :: | There was insufficient quantity or concentration of DNA loaded on the gel. Increase the amount of DNA, but don't exceed 50 ng/band. |
| :: | The DNA was degraded. Avoid nuclease contamination. |
| :: | The DNA was electrophoresed off the gel. Electrophorese the gel for less time, use a lower voltage, or use a higher percent gel. |
| :: | Improper W light source was used for visualization of ethidium bromide-stained DNA. Use a shortwavelength (254 nm) W light for greater sensitivity. Note: For preparative gels, using a longer wavelength (312 nm) W light will minimize DNA degradation. |
| If you see smeared DNA bands: | |
| :: | The DNA was degraded. Avoid nuclease contamination |
| :: | Too much DNA was loaded on the gel. Decrease the amount of DNA. |
| :: | Improper electrophoresis conditions were used. Do not allow voltage to exceed ~20 V/cm. Maintain a temperature <30° C during electrophoresis. Check that the electrophoresis buffer used had sufficient buffer capacity. This is done by checking the pH in the anode and cathode chambers. |
| :: | There was too much salt in the DNA. Use ethanol precipitation to remove excess salts, prior to electrophoresis. |
| :: | The DNA was contaminated with protein. Use phenol extractions to remove protein prior to electrophoresis. |
| :: | Small DNA bands diffused during staining. Add the ethidium bromide during electrophoresis. |
| If you see anomalies DNA band migration: | |
| :: | Improper electrophoresis conditions were used. Do not allow voltage to exceed ~20 V/cm. Maintain a temperature <30° C during electrophoresis. Check that the electrophoresis buffer used had sufficient buffer capacity. |
| :: | The DNA was denatured. Do not heat standards [except for l DNA/Hind III fragments (figure 1)] prior to electrophoresis. Dilute DNA standards in buffer with 20 mM NaCl. |
| FIGURE 1. The effect of ionic strength on the heat denaturation of l DNA/Hind III fragments. 500 ng DNA/Hind III fragments in NaCl concentrations of 0, 1, 2, 5, 10, 20, 30, and 40 mM were electrophoresed after heating at 65° C for 10 min. |
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