My biology lab experiments for DNA isolation and screening always shows pulling and smearing under the LED light. Me and my partner were pipetting very carefully not to disrupt the well, but the results were still unsatisfying. Was it because the agarose gel was not prepared currently? What can I do to avoid that from happening again? I would really like to have a clear result for my experiment. Please help. Thank you!
How are you making the agarose gel and what voltage are you running it?
It might be due to high running voltage. Make the gel with the proper percentage and run at 80 mv.
Possibly there is too much salt left in the dna. If you are using column technology then load less crude dna and after binding do 2 washes not just the one wash the kit recommends. Also make sure that the gel is completely cool before running the samples. Do not run the gel at more than 5volts/cm where the cm measurement is the distance in cm between the 2 electrodes of the gel system
I think you should adjust the voltage and timing it takes to run your gel. Be sure to always use a gel that is cool before carrying out the experiment.
1. Gel incompletely immersed in electrophoresis buffer.
Electrophoresis buffer should completely cover the entire gel during sample loading and run.
4.2. Low sample volume.
The sample or the ladder volume should be large enough to fill 1/3 of the total capacity of the well. Large wells
should not be used with small sample volumes. If needed the sample volume can be adjusted with 1X loading
dye.
4.3. Improper electrophoresis conditions.
Do not use an excessively high voltage for electrophoresis. Run the gels at 5-8 V/cm. To minimize band curving,
use a lower voltage for several minutes at the beginning of electrophoresis.
For fast electrophoresis under high voltage (up to 23 V/cm) use GeneRuler™ or O’GeneRuler™ Express DNA
ladders (#SM1551/2/3 or #SM1563, p.423).
To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value (which
is in many cases 5-8 V/cm, depending on the ladder) one has to:
– measure the distance between electrodes (cathode and anode) – X, cm.
– and multiply the X value by the recommended voltage (Y, V/cm)
– the result (X x Y) is the recommended voltage to be applied.
4.4. Bubbles or physical particles in the gel wells or in the gel.
Use pure water, clean flasks and clean equipment for preparation of gels.
Pour the gel slowly avoiding formation of bubbles. Bubbles can be removed with a pipette tip.
1. Gel incompletely immersed in electrophoresis buffer.
Electrophoresis buffer should completely cover the entire gel during sample loading and run.
4.2. Low sample volume.
The sample or the ladder volume should be large enough to fill 1/3 of the total capacity of the well. Large wells
should not be used with small sample volumes. If needed the sample volume can be adjusted with 1X loading
dye.
4.3. Improper electrophoresis conditions.
Do not use an excessively high voltage for electrophoresis. Run the gels at 5-8 V/cm. To minimize band curving,
use a lower voltage for several minutes at the beginning of electrophoresis.
For fast electrophoresis under high voltage (up to 23 V/cm) use GeneRuler™ or O’GeneRuler™ Express DNA
ladders (#SM1551/2/3 or #SM1563, p.423).
To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value (which
is in many cases 5-8 V/cm, depending on the ladder) one has to:
– measure the distance between electrodes (cathode and anode) – X, cm.
– and multiply the X value by the recommended voltage (Y, V/cm)
– the result (X x Y) is the recommended voltage to be applied.
4.4. Bubbles or physical particles in the gel wells or in the gel.
Use pure water, clean flasks and clean equipment for preparation of gels.
Pour the gel slowly avoiding formation of bubbles. Bubbles can be removed with a pipette tip.
The DNA bands can be seen by exposure of the gel to ultraviolet light, due to the the large increase in fluorescence of the ethidium bromide upon binding to the DNA. Agarose gels are submerged in electrophoresis buffer in a horizontal electrophoresis apparatus
Your DNA looks good. Smearing is usually observed in genomic dna so you don't have to worry about that.
Other than that the abnormal shape of your bands might be due to problem in your gel apparatus and that might be related to voltage output of the apparatus to the medium and gel. Try using other apparatus if you have and see if it changes anything.
Or it could be due to your personal mistake while loading the DNA.
If your DNA is not a PCR product, then I think there is nothing wrong with the DNA isolation result.
Hi, Salt and possibly overloading… i think you should load less DNA or dilute the sample
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