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
12 comments:
August 23, 2008 at 10:33 AM
Hello Liyanah,
Hi, ive got 1 question for you
1) Are there any precautions to take when handling ethidium bromide? If there is, can u state the precautions that should be taken?
Thanks!
From: Benjamin Ma
Class: tg01
0606181F
August 23, 2008 at 11:54 AM
Hello!
Got 2 questions.
Anw, why do you use TAE buffer because I use TBE buffer. Any differences? When going gel electrophoresis, will the voltage used affect how fast the DNA travels?
Thank you!
-Li Ping-
TG 02
August 24, 2008 at 7:02 PM
Hi Liyanah
1. May I know if the fragment size is bigger, the concentration of the agarose gel must be bigger or smaller?
2. What is the use of TAE buffer and ethidium bromide?
August 24, 2008 at 11:25 PM
Hi liyanah
Are there any advantages or disadvantages of using ethidium bromide?
What's the purpose of stuffing tissue at the mouth of the conical flask?
Thanks!
LeeJin
TG02
August 25, 2008 at 8:58 PM
to benjamin:
hey benjamin,
yes2, of course. because ethidium bromide is lethal, several safety measures have to be taken that is
1) it is to be done in the fume hood.
2) ensure that the right gloves are used and that is the nitrile gloves.
3) be careful not to have any form of contact with it although gloves are worn.
4) if spillage occurs, ensure that hands and any skin of contact is washed thoroughly with water and soap.
Liyanah Zaffre
0607718D
TG02
August 25, 2008 at 9:20 PM
to li ping:
Yes, there are differences, and we mainly use TAE buffer as it is useful for separation of smaller DNA fragments. Although TBE has a greater buffering capacity and will give sharper resolution than TAE buffer, it is more expensive and since we can use TAE buffer for the gel electrophoresis, we can use TAE instead of TBE, and save money. hehe.
and yes, the voltage do affect the rate of how DNA travels. The higher the voltage, the rate of seperation is faster as compared to lower voltage. However, the voltage and the time for it depends on the size of the gel and the fragment size. When the time is too long, may result in the fragments to migrate off the gel, and turning the voltage too high may damage the fragment or burn the gel itself.
Liyanah Zaffre
0607718D
TG02
August 25, 2008 at 9:44 PM
to sip:
erm, i think you forget to identify yourself. please do okay. :)
1)The concentration will affect the gel's pore size and thus, a low percentage of agarose gel will have a large pore size and will efficiently separate high molecular weight fragments. However, although a higher percentage is needed when the molecular weight of the fragment is small as in lower percentage, it will separate too much and will not turn out to be sharp bands.
2)TAE buffer is used as it provides faster electrophoretic migration of the DNA fragments, thus facilitate in the seperation while ethidium bromide is a fluorescent dye that is used for staining nucleic acids and thus, we used it for visualization purposes.
Liyanah Zaffre
0607718D
TG02
August 25, 2008 at 9:54 PM
to LeeJin:
hey LeeJin,the advantage of using ethidium bromide is for visualization purposes as it is a fluorescent dye. Without it, the bands wont appear. The disadvantage is that it is hazardous, thus many precautions need to be taken into consideration like i mentioned above.
The purpose of stuffing the tissue to the mouth of the flask is to prevent any evaporation which could affect the agarose gel.
Liyanah Zaffre
0607718D
TG02
August 26, 2008 at 3:28 AM
hey liyanah
since ethidium bromide is lethal, are there any alternative chemicals that can be used, which can also provide the same efficacy of staining the nucleic acids?
thanks
rusydiana
August 26, 2008 at 10:28 AM
Hi Liyanah !
I have some questions to ask you...
1)What is the mechanism by which nitrile gloves can protect your hands from being exposed to ethidium bromide? Also, I believe that nitrile gloves come in powdered and non-powdered forms, is there any difference between them in protecting the user?
2)How do you determine the percentage of your agarose gel based on the fragment size of you PCR products?
3)What does 1.5% agarose gel means?
4)What does electrophoresis buffer contains which helps it be able to maintain a constant pH?
5)What are the steps taken to quantitate your PCR products?
6)Why do you use a DNA ladder in your research application?
Thankz!
Han Yang
TG01
August 27, 2008 at 5:21 PM
Hi Rusydiana!
What I meant was not lethal. ethidium bromide is mutagenic. However, if proper precautions are taken, then it should be ok.
Alternative agent for staining is sybr-green. However, most labs do not use it cause of it's cost.
Liyanah Zaffre
0607718D
TG02
August 27, 2008 at 9:16 PM
hey han yang,
1)Nitrile gloves provide a strong barrier of protection and resist chemicals like solvents, greases and oils such that it doesnt easily penetrate to the skin unlike normal gloves. and yes, it does come in two forms, i'm pretty sure there are not much difference for both of them but what i think they are different in terms of comfort. non powdered gloves are actually more comfortable to use and more expensive.
2)Usually, we will have to refer to a table given to us, and there, it will state the concentration of agarose gel needed for a range of fragment sizes.
3)1.5% agarose indicates the concentration of the agarose gel, as it has varying concentration for different fragements and different concentrations would then affect the volume of TAE and ethidium bromide.
4) A buffered solution contains a mixture of a weak acid and its conjugate base. Thus, it maintains a constant pH by absorbing or releasing protons depending in the solution. For example, a small amount of strong acid introduced to pure water.
5) umm, in our lab, we usually dont quantitate our PCR product (check the DNA concentration). What we do, is the gel check and see if we have bands. If the bands come out thin, it would tell us that DNA concentration is quite low. therefore, we can estimate. but this step is not necessary for us.
6)It acts as a control and to also check if the bands come out according to the sizes.
Liyanah Zaffre
0607718D
TG02
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