Condition:瘦了3 公斤的懶人
Temperature: 25C
Background infromation: 請參考1,2,3,4,5 TRY~~~, 抱歉~~~太懶了~所以其它的沒po 上來
Discussion:
A higher concentration of agarose gel will reduce the DNA migration speed.
This could be because the higher concentration of agarose, there is less space between gel molecules, and the pores of the gel will be denser.
For a molecule smaller than the average pore size will be able to move relatively easily through the gel, whereas a resistance that depends on its radius will hold a molecule larger than the average pore size back. Therefore bigger DNA molecules (500bp) find it more difficult to get through the pore, and the migration speed is lower. The bigger DNA molecules (500bp) cannot travel very far in a concentrated gel, but for small piece of DNA a concentrated gel is rather good , because small molecules can pass through the pores, and the migration time is longer, therefore giving better resolution.
The increase of gel concentration didn’t give the better resolution for bigger DNA molecule (1000~500bp). From this data the best separation between 1000~500bp is under condition 3.2% of agarose gel, but this gel is also run for the longest time. To have better separation in bigger DNA molecules (1000~500bp) it would be interesting to see a 1.1 % gel for longer than was done in this experiment.
From the photograph, that is difficult to see each band in 20bp DNA ladder for bigger DNA molecules ( 540bp), the bands are too close. For smaller DNA molecule (≦500bp), the separations are better the gel concentration increases.
By comparing the voltage and time taken, it can be seen that the DNA ran quicker in higher voltage. This is due to the higher the applied voltage, the larger the field strength across the medium, and the faster will a molecule migrate. So increasing the applied voltage will cause the DNA molecules to migrate more quickly. This can be an advantage, as it saves time and reduces diffusion of the DNA migrating.
From comparing the distance, the separation of bigger DNA molecules is greater in lower voltage. This could because at the lower voltages, it takes a longer time for DNA molecule to travel, therefore giving better resolution.
But higher voltage also increases the heat produced. The temperature may affect the result. The diffusion of migrating zones of charged molecules increases with an increase in temperature. If the electrophoresis takes several hours to run, diffusion effects can become very significant. When cooling is inadequate, those parts of the migrating zones in the warmer part of the gel will move faster than those in the cooler part. This unequal speed of migration results in curved bands, which can lead to overlap between neighboring zones, and consequently very poor resolution. This situation happened in some gels in this project.
This rsult showing the separation between small DNA molecules is better in high voltage. For the small DNA molecules, the less molecular weights, and molecules travel further down to the gel easily and quickly with higher power supply, therefore better resolution.
To compare and contrast 3 gels run under the same condition, the only variation is the time difference. Two of the gels reached to the same place in the approximately time; this means the experiment can be repeated.
By comparing the distance, the longer the electrophoresis, the further that fragments migrate, and the greater the separation between each band. However some bands of small DNA molecules may run off the gel. From the result, for DNA molecules 600bp, the bands were still difficult to distinguish from the photo, especially between every 20bp.
The difficulties found in this method:
The gel can break easily during staining especially at low agarose concentration. To avoid this gels can be stained on the tray, but destaining will take longer, due to having less access to the gel.
The gel stained with less concentrated ethidium bromide; reflected less during taking photos. Some lab may run electrophoresis with ethidium bromide inside the gel. There are two problems with that: ethidium bromide binds to DNA molecule, and change the mobility of fragments at high DNA concentration, thus decreasing the reliability of fragment-size measurements. Some of the ethidium bromide may be lost as it travels down the gel, therefore there is still need to stain again after. Also for safety reason, it is better to use as little ethidium bromide as possible. Therefore it is concluded that it is best to stain the gel after the electrophoresis.
The gel is very fragile, and difficult to handle when the concentration is too low, and takes more time for gelling, sometimes breaks easily during staining.
When the concentration is high, the Metaphor agarose powder is more difficult to dissolve; because of the energy needed to melt an agarose gel increases as the gel concentration increases, and gelling is too quickly, easily to form lumps.
The suggested minimum concentration of Metaphor agarose gel from the BioWhittaker Molecular Applications website is 1.8%. However, it was found that lower concentration were possible.
The determination of time is difficult, because the dye will change the migration speed under the different concentration of agarose gel.
The following table is a migration table of double-stranded DNA in relation to Bromophenol blue (BPB) and Xylene cyanol FF (XC) in Metaphor Agarose gels. (http://
www.bmaproducts.com/technical/electrophoresisofdnainagarosegels.shtml)
1X TBE Buffer
% AGAROSE XC BPB
2.0 310 40
3.0 140 35
4.0 85 30
5.0 60 15
It is difficult to measuer the sharpness and how narrow is the band by looking at the photographs. But by comparing the result of different concentration of agarose, the high concentration seems to give a narrower band then the low concentration. This could because of the higher concentration give denser gel, fragments are difficult to migrate, also difficult to diffuse, and therefore the bands are sharper and narrower.
The best result for all the bands (1000 ― 40bp) in this project is provided in the photograph as above
Suggested methods:
This experiment tried to find an easy way to demonstrate the DNA fingerprinting.
From the result, this method is suitable to use when the samples are small DNA molecules (500bp) in this project, but that will be interesting to try the gel run under low concentration of agarose.
For good separation in locus D1S80 which the range size is from 369―801 base pairs is difficult by using this method, there are no significant result showing good resolution in all of this range. At the D1S80 locus, most individuals have alleles containing between 14 and 40 repeats, to be able to determine each alleles from the electrophoresis, it requires a good separation of each band with 20bp DNA ladder. The most likely conditions to give good results in this range are under 2.5 Metaphor agarose gel; 10v/cm stop as Xylene Cyanol reached the end of the gel.
呼~~ 好一陣子可以不用掙扎了~~~哈~~哈~~
