PCR Practice: Create forward and reverse primers that amplify the majority of the sequence below. Draw...

PCR Practice:

Create forward and reverse primers that amplify the majority of the sequence below. Draw circles around the section of the sequence your primers are taken from. Highlight the region of the sequence that your PCR will amplify. Indicate the 3’ and 5’ ends on your primers.

>gi|297828897|ref|XM_002882285.1| Arabidopsis lyrata subsp. lyrata glyceraldehyde-3-phosphate dehydrogenase C subunit (GAPC), mRNA

CTCCACGTTCTTCTCTCTTTTAAATAGACCCTTCACGGACCCTTCTCACTCACCTATCTCACTGTTAAAT

ATCTCTCTGTGAATCTCATCTTCAACCTCTCTTACACACTCGCGTTTTCGATTCAAACAATGGCTGACAA

GAAGATTAAGATCGGAATCAACGGATTCGGAAGAATCGGTCGTTTGGTTGCTAGAGTTGTTCTTCAGAGG

GACGATGTTGAGCTCGTTGCTGTTAACGACCCCTTCATCACCACTGAGTACATGACCTACATGTTCAAGT

ATGACAGTGTTCACGGTCAATGGAAACACAATGAACTCAAGATCAAGGATGAGAAGACCCTTCTCTTCGG

TGAGAAGCCAGTCACTGTTTTCGGCATCAGGAACCCTGAGGATATCCCATGGGCCGAGGCTGGAGCTGAC

TACGTTGTTGAGTCTACCGGTGTCTTCACTGACAAGGACAAGGCTGCTGCTCACTTGAAGGGTGGGGCCA

AGAAGGTTGTCATCTCTGCCCCCAGCAAAGACGCACCCATGTTTGTTGTTGGTGTCAACGAGCACGAATA

CAAGTCCGACCTTGACATTGTCTCCAACGCTAGCTGCACCACTAACTGCCTTGCTCCCCTTGCCAAGGTT

ATCAACGACAGGTTTGGAATTGTTGAAGGTCTTATGACAACAGTCCACTCTATCACTGCTACTCAGAAGA

CTGTTGATGGTCCATCAATGAAGGACTGGAGAGGTGGAAGAGCTGCTTCATTCAACATTATTCCCAGCAG

CACTGGAGCTGCCAAGGCTGTCGGAAAGGTGCTTCCAGCCCTTAACGGAAAGTTGACCGGAATGTCTTTC

CGCGTCCCAACCGTTGATGTCTCAGTTGTTGACCTTACTGTCAGGCTCGAGAAAGCTGCTACCTATGATG

AAATCAAAAAGGCTATCAAGGAGGAATCTGAAGGCAAACTTAAGGGAATCCTTGGATACACCGAGGATGA

TGTTGTCTCAACTGACTTCGTTGGTGACAACAGGTCGAGCATTTTTGACGCCAAGGCTGGAATTGCATTG

AGCGACAAGTTTGTGAAATTGGTGTCATGGTACGACAACGAATGGGGTTACAGTTCCCGAGTGGTCGACT

TGATCGTTCACATGTCAAAGGCCTAAGCTAAGAAGCAGATCTCGAACGGCGAGGAGTGGAAAGTCATCTG

TTCATCCTCTTTTATGGTCTGACTTTGTCGTTTTCGAATAAAATTTCTTTGAACTTGGAACCCTTTTTTT

TTTTTTTTGGTTTTCTTAATTCTCATTCATGTGAGTTGATGGGAGTTTGTAGACCGGTGTTTTACTGAAG

CCCTTTCGTTTTTGGCTTTTGATATATTGAGTTACTTATGGTTTTTCATTTTGTTTCACTTCTCTTTTTT

TCTATATACTAAATCAAATCTGAACGAAC

Forward:_____________________________________________

Reverse:_____________________________________________

- Primers must be 18-30 base pairs in length

- Cannot contain intra-complementary sequences

- Must have a G-C clamp of 2-5 base pairs

- Also include GC content of primers and melting temperature

- Include number of nucleotides in primer

- G/C clamp- write out the G/C clamp in this format: 3' - GCC (whatever the G/C clamp is should go in the place of the GCC)

- Circle the template strand in the location the primers are taken from

Expert Solution

The DNA has two strands - one is 5' to 3' which is the coding sequence and the other is 3' to 5' which is the template strand. The forward primer is 5' to 3' taken as it is from the sequence which is also 5' to 3'. The forward primer binds to the template strand from the begining which is 3' to 5'. The reverse primer binds to the coding strand from the end. The reverse primer is built in 5' to 3' direction by reversing the end part and complimentary to it.

Forward primer-

5'- CAACCTCTCTTACACACTCGCG- 3'

Tm of the primer= 59 degree celsius

Length of the primer = 22

GC content of the primer is = 55%

Reverse primer-

5'- ATGACTTTCCACTCCTCGCCG- 3'

Tm of the primer= 60 degree celsius

Length of the primer = 21

GC content of the primer is = 57%

The primer will attach to the bold and underlined part-