13. You want to amplify the cDNA made from mRNA of gene “X”. The 5’ and 3’ end o

Question

13. You want to amplify the cDNA made from mRNA of gene “X”. The 5’ and 3’ end of the cDNA sequence is shown below. You have designed two primers which you think can amplify cDNA made from gene “X” and which are complementary to the bold sequences (also shown below). These two primers are 15 nucleotides in length.

Gene X: 5’-GCTGATCGATGCGATAGGA...........CGTGACTGACGAGCTGACT-3’

b – You start your PCR reaction (denaturation: 95°C, 1 minute; annealing: 60°C, 1 minute; extension 72°C, 1 minute; for 35 cycles). But at the end of the reaction, when you analyze your PCR product you see that nothing has amplified.
- Why did your reaction not work? How would you correct this so that you have correct amplification in the next experiment?

Explanation / Answer

Gene X: 5’-GCTGATCGATGCGATAGGA...........CGTGACTGACGAGCTGACT-3’

Therefore the two primers will be:

Primer 1--> 3'-CGACTAGCTACGCTA-5' Primer 2-> 3'TGACTGCTCGACTGA-5'

The annealing temperature used in the PCR reaction is 60°C. However, that temperature is too high and will not allow the two primers and the cDNA to base pair with each other fopr proper annealing.

The proper annealing temperature for each set of primers with its complementary sequence using the formula :

4(G+C) + 2(A+T) --> Here it is = 4(8) + 2(7) = 32+ 14 = 46

TYhus the annealing temperature in this reaction should be 46°C or just a little higher for proper annealing.

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