Hello my dearest friend, I hope all of you are well. It was great meeting up during the campus discussion. :)
Anyway, as for this week, I will continue from where I stop in the previous post.
After primer optimization is done, we then need to visualize the PCR products to determine at which temperature is the best. Thus, we need to load the product into the agarose gel, and subject it to gel electrophoresis.
How to make an agarose gel?
Depending on the fragment size, the percentage of the agarose gel differs. As for me, my fragment size is about 1.2kb and thus I use the 1.5% agarose gel.
Methods to make a 1.5% agarose gel
1. Select the suitable agarose gel mould and the combs, then place it in the fume hood.
2. Ensure that the mould is level, by using the air bubble gadget (air bubble must be at the centre of the gadget).
3. Calculate the amount of agarose, volume of the TAE buffer and ethidium bromide needed.
Calculation of the amount of agarose and volume of TAE buffer
For 1.5% (wt/vol) of agarose gel, we need 1.5g of agarose in 100ml of TAE buffer.
Since the suitable mould can only hold up to 60ml, we only need 60ml of TAE buffer.
100ml -> 1.5g of agarose
60ml -> 1.5/100 x 60 = 0.9g of agarose in 60 ml of TAE buffer.
Since the suitable mould can only hold up to 60ml, we only need 60ml of TAE buffer.
100ml -> 1.5g of agarose
60ml -> 1.5/100 x 60 = 0.9g of agarose in 60 ml of TAE buffer.
Calculation of volume of ethidium bromide needed
Initial concentration of ethidium bromide: 10mg/ml
Final concentration needed for agarose gel: 0.5 ug/ml
Therefore, using M1V1=M2V2
10000ug x V1 = 0.5ug x60ml
V1= 0.5 x 60 / 10000 = 0.003ml = 3ul of ethidium bromide
4. Weigh out 0.9g of agarose powder and add it into a dry conical flask.
5. Obtain 60ml of TAE buffer using a measuring cylinder.
6. Pour the 60ml of TAE buffer into the conical flask.
7. Stuff tissue at the mouth of the conical flask and place it in the oven for 1 minute.
8. At intervals, take out the conical flask and swirl it, to check if the agarose gel has melted.
9. After which, the agarose gel solution should be clear and colourless and let the solution is cool for a few seconds.
10. Add in 3ul of ethidium bromide to the solution and swirl it again.
11. Pour the agarose gel solution to the mould and place the combs in their respective positions.
12. The gel is then left to harden and solidify for about 10-15 minutes.
Why use agarose instead of polyacrylamide?
Agarose gel is mainly used to separate smaller molecules like nucleic acids eg DNA, while polyacrylamide are capable and often used for separating larger molecules like proteins. Also, polyacrylamide gel is more expensive than that of agarose and thus, not used in this case as not necessary.
Gel electrophoresis
1. Place the agarose gel mould with the solidified agarose gel into the electrophoresis tank containing the TAE buffer. Make sure the whole gel is immersed in the buffer.
2. Add 2ul of loading dye into each of the tubes of the PCR products. Spin it down to mix it well.
3. Pipette out 10ul of the resultant mixture and load them into the respective lanes.
4. Set the electrophoresis at 120 voltage for 20 minutes, and ensure it is running.
Principle of gel electrophoresis
- Gel electrophoresis is used to separate macromolecules eg proteins or DNA that might differ in size, charge or conformation.
-When the gel is run, the molecules (eg protein in particular) will migrate towards the positive (anode) or negative (cathode) depending on their charge.
- Thus, nucleic acids foe example DNA has a negative charge due to the phosphate group attached, will migrate towards the anode.
- As noted from the methods, an electrophoresis buffer is used. Why? It is because the buffer provides ions to carry a current and also to maintain a constant pH.
Once that is done, I would then proceed on to do the gel check, to see my PCR products that will be seen as bands. For this step, a machine also known as Gel Doc is used. As this step is after primer optimization, we need to know the right temperature for the primer. The right temperature would show bright and sharp bands.
Examples
Final concentration needed for agarose gel: 0.5 ug/ml
Therefore, using M1V1=M2V2
10000ug x V1 = 0.5ug x60ml
V1= 0.5 x 60 / 10000 = 0.003ml = 3ul of ethidium bromide
4. Weigh out 0.9g of agarose powder and add it into a dry conical flask.
5. Obtain 60ml of TAE buffer using a measuring cylinder.
6. Pour the 60ml of TAE buffer into the conical flask.
7. Stuff tissue at the mouth of the conical flask and place it in the oven for 1 minute.
8. At intervals, take out the conical flask and swirl it, to check if the agarose gel has melted.
9. After which, the agarose gel solution should be clear and colourless and let the solution is cool for a few seconds.
10. Add in 3ul of ethidium bromide to the solution and swirl it again.
11. Pour the agarose gel solution to the mould and place the combs in their respective positions.
12. The gel is then left to harden and solidify for about 10-15 minutes.
Why use agarose instead of polyacrylamide?
Agarose gel is mainly used to separate smaller molecules like nucleic acids eg DNA, while polyacrylamide are capable and often used for separating larger molecules like proteins. Also, polyacrylamide gel is more expensive than that of agarose and thus, not used in this case as not necessary.
Gel electrophoresis
1. Place the agarose gel mould with the solidified agarose gel into the electrophoresis tank containing the TAE buffer. Make sure the whole gel is immersed in the buffer.
2. Add 2ul of loading dye into each of the tubes of the PCR products. Spin it down to mix it well.
3. Pipette out 10ul of the resultant mixture and load them into the respective lanes.
4. Set the electrophoresis at 120 voltage for 20 minutes, and ensure it is running.
Principle of gel electrophoresis
- Gel electrophoresis is used to separate macromolecules eg proteins or DNA that might differ in size, charge or conformation.
-When the gel is run, the molecules (eg protein in particular) will migrate towards the positive (anode) or negative (cathode) depending on their charge.
- Thus, nucleic acids foe example DNA has a negative charge due to the phosphate group attached, will migrate towards the anode.
- As noted from the methods, an electrophoresis buffer is used. Why? It is because the buffer provides ions to carry a current and also to maintain a constant pH.
Once that is done, I would then proceed on to do the gel check, to see my PCR products that will be seen as bands. For this step, a machine also known as Gel Doc is used. As this step is after primer optimization, we need to know the right temperature for the primer. The right temperature would show bright and sharp bands.
Examples
L=ladder, Temperatures: 55oC-65oC,
Best temperature: Lane 5 = 58oC
Why? Lane 1-4 shows non-specific binding. This is due to too low annealing temperatures. When that happens, the primers don’t anneal properly and thus will lead to non specific products as seen.
Lane 8-12 , although the bands are pretty bright, they are not as sharp as Lane 5.
(Sorry, pictures are not that clear so can’t really visualize the minute differences)
2nd example
As for this, there are actually 12 lanes and the ladder. However Lanes 1-6 shows smearing and non specific binding. And Lane 7 shows faint band as compared to Lane 8. Thus, it is pretty obvious in this case that Lane 8 is the best temperature. For this also, Lane 9-12 do not show any bands. This is because the temperatures are too high for the primers to anneal and thus, no products.
After which, when we have chosen the best conditions for PCR to run, we can then proceed on to do PCR for the different DNA samples at that condition. This again, will be explained in my future postings.
Alrighty, till next time then. Take care lovelies. :)
Liyanah Zaffre
0607718D
TG02
