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Sample InjectionThe amount of sample injected into the capillary along with the narrowness of the separation band determines the height of the signal measured for each base. To have the best signal to noise we would normally want the maximum amount of sample to be injected that does not produce a signal that saturates the detection system. The amount of sample injected is determined by the product of the [injection voltage] x [the length of time the voltage is applied]. If the amount of sample injected is doubled the area under the signal curve will double but the height will not. In fact the signal height may only increase by about 30 %.
It has been found that the injection voltage should be limited to about 1.5 kV [25 volts/cm] for a 60 cm capillary. The reason for this limitation is that the sample and capillary form a series circuit and the voltage in this circuit will be highest in the area of highest resistance. If the sample is injected from de-ionized water which has a very high resistance compared to the buffer in the capillary it will therefore have a larger voltage drop at the entrance to the capillary. This voltage drop can be a source of air bulbs at the tip of the capillary. Air bulbs that enter the capillary will produce erratic and declining capillary current. The width of the injection plug is determined by the voltage and the length of time of the injection. If the voltage is 25 volts/cm then the maximum recommended time is about 80 seconds. After this time the width of the injection plug becomes a factor and affects the band width of the DNA separation and reduces resolution.
Sample preparation can affect how much sample is actually injected. If the samples contain different amounts of salt then different amounts of sample will be injected. As the salt content increases in a sample the electrical resistance decreases. If the electrical resistance of the sample decreases then the electrical field [volts/cm] in the sample is less during the injection. The DNA in the sample is forced into the capillary by the electrical field that it is subject to in the sample. If the volts/cm in the sample is less, the DNA will move at a slower rate into the capillary. The amount of template in the sample also can affect the amount of sample injected. Template can plug the entrance of the capillary and prevent DNA from entering. It can also create regions of high electrical resistance which can lead to air bulb formation and current failure in a capillary later in a run. Removing the template or using small amounts will help reduce this problem.
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