Your crazy cousin Bob loves to do science, but has some serious technical issues

Question

Your crazy cousin Bob loves to do science, but has some serious technical issues. He did PCR on three days last week and ran separate gels for each of his products. He shows them to you, swearing he followed the protocol exactly each time. To help Bob figure out where he went wrong, you did the PCR reaction and it worked perfectly. Make a hypothesis of what could be wrong with EACH of Bob’s experiments AND explain to Bob the rationale for each of your theories/what he should do to improve his results.

Explanation / Answer

If your gel is correct, the following troubleshooting will be applicable for each trail of Bob,

Trail 1 gel resembles your gel result, but the product concentration is too less. Here, Bob would have added either insufficient enzyme or primers and the primer annealing temperature may not be appropriate/optimum to get the target amplification at the maximum rate. So that, he needs to standardize the enzyme or primers concentration that required for the maximum amplification of the target in fixed PCR cycles. If the problem is temperature, he needs to run temperature gradient PCR (with reference to Tm of the primers +/- 5oC) that would provide optimum temperature for target amplification.

Trail 2: Here, it seems that he did not load his product or ladder on the gel. There are no bands on the gel.

Trail 3: He would have added different primers instead of target gene amplification primers so that, he got an intense band at the different length. Maybe the PCR condition used by Bob are optimum for the different primer amplified target instead of his actual target that shown in your gel. So, change the primers, use its specific Tm+/- 2oC, sufficient concentrations of primers and enzyme and PCR cycles to get actual product.   

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