In the above gel, First we loaded ladder (”L”on the gel) which is 1 kb (1000 bp)

Question

In the above gel, First we loaded ladder (”L”on the gel) which is 1 kb (1000 bp) gives you the molecular size of each bands that we can use as a marker.

Then DNA/sample is loaded in all wells. If we want to know if we have that particular DNA band of interest in the gel , 1) we need to know the molecular size of the DNA and read the band comparing to the ladder bands

OR

2) we need to have a positive control (“P” on the gel) to match our sample DNA to the control DNA, if both of the DNA bands (control and sample) aligns at the same patterns, then we can also confirm that we have our DNA of interest in the gel. The control can have one or multiple bands depending on the insertion.

3) We can also have a negative control (“N” on the gel) which did not show any bands that corresponds with all samples that do not have that specific DNA band. It is also an authentication of the gel that our samples did not have contamination because usually we load the negative control that comprises all reagents used in other sample tubes excluding the DNA or distilled water.

Examples how can we record the results of our gel and all the visible bands:

1. One band in positive control: + Samples can be   + or -

3. Two bands in positive control: +/+

The samples can be   +/+   OR      +/-   OR   -/+   OR    -/-

Question for practice:

1. Use this example and check/practice how you will record the results from the above gel?

2. We visualized 3 bands for the DNA in positive control and few samples showed 2 bands and few showed 3 bands and some showed none, how would you record the results?

1 kb 500 bp 300 bp 400 bp. L 1 2 33 P N Fig.1. Agarose gel electrophoresis of semi-nested RT-PCR for the N gene of bovine coronavirus (BCoV). L 100bp DNA ladder; 1-3: examples of bovine fecal positive samples; P positive control; N negative control (DEPC-treated ultra pure water). Specific amplicons are 306-base-pairs long. Picture Source: http://www. arttext&pid-S0100-736X2009001100001;

Explanation / Answer

1. Use this example and check/practice how you will record the results from the above gel?

SInce the postive control has three bands (at 300 bp, below 500 bp and below 1 kb), postive control can be recorded as

postive control : +/+/+

Since the samples 1 - 3 has one band at 300bp, they can be recorded as follows

Sample 1 : +/-/-   

Sample 2 : +/-/-

Sample 3 : +/-/-

Negative control : -/-/- (since the negative control has no band )

2. We visualized 3 bands for the DNA in positive control and few samples showed 2 bands and few showed 3 bands and some showed none, how would you record the results?

Since 3 bands are visualized for the DNA in positive control

Positive control: +/+/+

samples which showed 2 bands

Sample : +/+/-

samples which showed 3 bands

Sample : +/+/+

samples which showed no band

Sample: -/-/-

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