Based on the size you determined for your PCR product in the previous quesion (2

Question

Based on the size you determined for your PCR product in the previous quesion (244 bp), theoretically how long of a polypeptide could be encoded in the PCR product (assume there are no introns). What would be the approximate molecular weight (in kilodaltons) of the polypepide (average size of an amino acid = 111 Daltons)? SHow all work please.

WHat do you predict would happen to the results of the PCR if you lowered the annealing temperature by 15 degrees C? (First annealing temperature was 39 degrees C)

may be needed to answer the question

You are given the following 450 bp DNA primers that anneal to the shaded sequence. Design P regions of the sequence below. 10 pts total. G TCGACCCAC GCGTccGCAA CACCACGcCA AACTACCACC ACCACAACTA cCACAACTAC AACCAcTTCA ACTAATTcTA cTTCAAATAA ATTACCAAAT AAT GGTGATA ATACTGTATO ATTTGATGAA AAATTOGATO CAAACGTATC TCATTCAAAT TTAAATGATG ATTATAAAG TTCAGGTAGT TCTGATTCAC CAA ATA TAGATTAA AT AC GTCAAA GATTAAAAAT GTATAA ACAA AAA rcTGAAA AAAGATTAAG TCATAGACCA AAATCAATTA AATTAGAACA TAAAAAGAAT GTTTTAGGTG AAATTATTTA TAAAGGACAT cCAAGTTGGG CATTAATGTT AAATAT TCAA ACTGGTATTA GAAATGcGGT TGGTAAATCA. AT GGGTACAG AAGGAGGAGT TAATGCAAAG ACAC AGTATCATAC a) write the primer sequences below. Be sure to label the 5- and 3-ends of each sequence. Forward Primer s TO TAA TTTATG 21 TTGTG 3 Reverse Primer: b) Give the approximate Tr of each primer show your work: c) What PCR annealing temperature would you use with your primers? Briefly explain why Tinn armaung C. This than the ermprotone token S degres Less d) what is the total size of base pairs) of the expected PCR product? Product is att bp

Explanation / Answer

Assume there are no introns and stop codon and translation starting from the beginning of 244 bp to last there would be 244/3 triplet codon for amino acids i.e. 81 amino acids with molecular weight 111 Dalton each. This equals to 111*81 or 8991 Dalton or 8.991 Kilo Dalton.

If you lowered the annealing temperature by 15 degrees C from first annealing temperature of 39 degrees C the primer will non-specifically bind to other sites as well and will produce additional combination of PCR products. At temperature near to melting the specificity increases and at lower temperature the primer mis-align to other relatively low complementary stretches.

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