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In: Biology

After PCR reaction has taken place, what would be the estimated size of the product (not...

  1. After PCR reaction has taken place, what would be the estimated size of the product (not inserted into cloning vector).

Equation for product length: Product length = (Position of antisense primer-Position of sense primer) + 1

  1. Based on your expected product size, draw an agarose gel depicting a DNA ladder, amplified product and negative control (amplification did not work). To draw an agarose gel, use power point. Use black for agarose background, white for DNA and ladder. Remember to label all the wells. Describe components added for PCR product-negative control (be creative), assume the same PCR parameters.

Solutions

Expert Solution

However, it does depend on the size of the bands. If the bands for the negative control show products much smaller than the samples or positive control, it could possibly be primer dimer. But, if the band is the same or similar size as the positive control, it probably means there is some template contamination. Any one of the reagents could be contaminated, or it could be the pipetter or tips as Taras suggested.

The simple solution would be to re-setup the reaction using new reagents. If you want to find the source of the contamination, then you would need to change only one reagent at a time and set up a series of identical reactions where one reagent was change in each one. Filter tips could eliminate the possibility that the pipetter is the source of contamination. But you may also get by by cleaning the pipetter well since filter tips are more expensive.

gladiator answered 2 years ago
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