5) Focus on the gel below. This gel has 2 major problems:1) the smearing through

Question

5) Focus on the gel below. This gel has 2 major problems:1) the smearing throughout the lanes, and 2) the lack of a distinct PCR product. Wbat accounts for the smearing in lanes 2, 3, and 4? There are some common mistakes that account for can cause smearing of the PCR products. One is that the DNA sample was contaminated with a protein that causes smearing Lanes 6 & 8 do not have any bands corresponding to amplicons. If these were your results, what are 2 changes you would make to your PCR reactions? 5 6 78

Explanation / Answer

Smearing in lane 2,3 and 4 is due to there maybe a call if too much sample is loaded on the gel.

Gel run too fast at high voltage can also have smeared band.

Poorly polymerised gel is also the case.To ensure sharp banding use fresh made APS and TEMED at least 30 minutes before so that polymerisation takes place accurately.

Particulate in the sample: Particulate can be removed by centrifugation prior to loading on the gel.

1 When DNA sample is contaminated with protein it causes smearing pattern.

2 Similarly when RNases treatment is not given before loading it also leads to smearing pattern.

The two changes that can be made to the PCR reaction are:

1.The annealing temperature could be too high due to which the annealing wouldn't have taken place.

2.There can be impurities present in the loading sample like protein and RNA which has to removed prior to the loading of sample by RNases and proteses treatment.

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