Phase 2 – Agarose Gel Electrophoresis
1. Obtain our four H, E, L, and P labeled micro tubes and place them on ice.
2. Set the micro pipet to 2.0 ul.
3. Transfer 2.0 ul blue dye to each of the four H, E, L, and P micro tubes.
4. Mix the four micro tubes by using this two-step method: * Place each tube briefly on the vortex to get the components to collect at the bottom of the micro tubes * Pulse spin the four micro tubes in the centrifuge to mix all of the components completely.
5. Obtain the DNA marker and label it “M”
6. Heat all the samples at 65 degrees Celsius for five minutes, then replace them on ice.
7. Obtain Agarose Gel
8. Pull apart gently the ends from the casting tray and remove the combs from the gel.
9. Place the casting tray onto the central platform in the gel box.
10. Making sure that the wells at the negative end of the box.
11. Pour slowly in about 275ml of electrophoresis buffer into chamber until the gel is submerged.
12. Pipet 10 ul from each of the four micro tubes M, H, E, L, P and instert them into the agarose gel following the table below:
Lane Sample
1 M
2 L
3 P
4 E
5 H
13. Slide a cover over the …show more content…
Lambda comes from a bacteriophage, but is harmless to humans and other eukaryotic organisms. Lambda has approximately 48,000 base linear pairs. Three common restriction enzymes: EcoRI, HindIII and PstI are used to separate the DNA fragments. The task at hand was to separate the fragments of DNA. Separation was to be done based upon size using gel electrophoresis. Then compare the DNA fragment of known sizes by millimeter or molecular weights, or standards to the DNA restriction fragments. Each enzyme cuts at a different place in the lambda DNA. This happens because each enzyme has a different restriction fragment that would produce different sizes. In this activity our standard is the HindIII digest of lambda DNA, which is frequently used as a DNA molecular weight
1. Obtain our four H, E, L, and P labeled micro tubes and place them on ice.
2. Set the micro pipet to 2.0 ul.
3. Transfer 2.0 ul blue dye to each of the four H, E, L, and P micro tubes.
4. Mix the four micro tubes by using this two-step method: * Place each tube briefly on the vortex to get the components to collect at the bottom of the micro tubes * Pulse spin the four micro tubes in the centrifuge to mix all of the components completely.
5. Obtain the DNA marker and label it “M”
6. Heat all the samples at 65 degrees Celsius for five minutes, then replace them on ice.
7. Obtain Agarose Gel
8. Pull apart gently the ends from the casting tray and remove the combs from the gel.
9. Place the casting tray onto the central platform in the gel box.
10. Making sure that the wells at the negative end of the box.
11. Pour slowly in about 275ml of electrophoresis buffer into chamber until the gel is submerged.
12. Pipet 10 ul from each of the four micro tubes M, H, E, L, P and instert them into the agarose gel following the table below:
Lane Sample
1 M
2 L
3 P
4 E
5 H
13. Slide a cover over the …show more content…
Lambda comes from a bacteriophage, but is harmless to humans and other eukaryotic organisms. Lambda has approximately 48,000 base linear pairs. Three common restriction enzymes: EcoRI, HindIII and PstI are used to separate the DNA fragments. The task at hand was to separate the fragments of DNA. Separation was to be done based upon size using gel electrophoresis. Then compare the DNA fragment of known sizes by millimeter or molecular weights, or standards to the DNA restriction fragments. Each enzyme cuts at a different place in the lambda DNA. This happens because each enzyme has a different restriction fragment that would produce different sizes. In this activity our standard is the HindIII digest of lambda DNA, which is frequently used as a DNA molecular weight