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1. Calculate the Tm for the following oligonucleotide primers:
5' CAT CTG CAC AAA ATC GGC 3' Tm= 54 C
5' GTG GCC CAT TTC GTG GTG 3' Tm= 58 C
2. Write out the sequences which are complementary to the following DNA strand.
5' GTC GAG ATC TGA GGC GCA 3'
3' CAG CTC TAG ACT CCG CGT 5' Antisense primer
Fill out this "order sheet" to synthesize the complementary oligonucleotide to the strand above.
5' TGC GCC TCA GAT CTC GAC 3'
3. A researcher wants to amplify the renin gene sequences shown below (+50 to +140). Underline two sequences on the correct strand that could be used as 20
nucleotide PCR primers. (The numbers above the sequence indicate the position in the cDNA.) Watch out for internal palidromic sequences!
+50 +95
5' ACT GGC GCC AGG AGG ATG CCT CTC TGG GCA CTC TTG TTG CTC TGG 3'
3 TGA CCG CGG TCC TCC TAC GGA GAG ACC CGT GAG AAC AAC GAG ACC 5'
+96 +140
5'ACC TCC TGT AGC TTC AGT CCT CCG ACA GAG CGG GGA GTA AAA AAA 3'
3'TGG TGG ACA TCG AAG TCA GGA GGC TGT CTC GCC CCT CAT TTT TTT 5'
A seq for sense primer - ( 5' TCTGGGCACTCTTGTTGCTC 3' )
A seq for antisense primer - ( 3' GCTGTCTCGCCCCTCATTTT 5' )
4. Calculate the expected length of a PCR product amplified from a cDNA using 20 nucleotide primers which anneal to the sense strand from + 169 to +189 and
the antisense strand from +834 to +854.
No PCR product will be made (Obp). A product of 685 bp will be made if the order of the primers is reversed.
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