In lab 6, you will set up a PCR reaction to yield amplified DNA products of the rpo B(RNA polymerase B) and 16s rRNA genes. In lab 7, you will set up a sequencing reaction using about 40 ng of these PCR products that you will then add a sequencing primer to the sequencing reaction. If a sequencing reaction is set up with a 100-fold excess of primer to the PCR-amplified DNA product you want to sequence, calculate the number of sequencing primers you will need to add to this sequencing reaction. To perform this calculation, assume your 40 ng sample of double stranded DNA is 4,000 base-pairs (bp) long. Using these two variables of your PCR product [length (in bp) and amount (in g, grams)], you can calculate the number of DNA molecules being sequenced with two conversion factors using the formula below: The average weight of 1 bp is approximately ~650 (g/mole)/bp Avogadro’s number which is 6.02 X 1023 molecules/mole The easiest way to solve this problem is to figure out how much one molecule of DNA would weigh. Then take your 40 ng sample of DNA and divide it by the mass/molecule and then multiply by 100 (excess primer).
Q5. In lab 6, you will set up a PCR reaction to yield amplified DNA products of the rpo B(RNA polymerase B) and 16s rRNA genes. In lab 7, you will set up a sequencing reaction using about 40 ng of these PCR products that you will then add a sequencing primer to the sequencing reaction.
If a sequencing reaction is set up with a 100-fold excess of primer to the PCR-amplified DNA product you want to sequence, calculate the number of sequencing primers you will need to add to this sequencing reaction.
To perform this calculation, assume your 40 ng sample of double stranded DNA is 4,000 base-pairs (bp) long.
Using these two variables of your PCR product [length (in bp) and amount (in g, grams)], you can calculate the number of DNA molecules being sequenced with two conversion factors using the formula below:
The average weight of 1 bp is approximately ~650 (g/mole)/bp
The easiest way to solve this problem is to figure out how much one molecule of DNA would weigh. Then take your 40 ng sample of DNA and divide it by the mass/molecule and then multiply by 100 (excess primer).
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