Posts Tagged ‘primers’

Question by coastcutie: WHat 4 nucleotide long primers could the scientist use to PCR amplify the Pme1 cut DNA?

Best answer:

Answer by Oops!
this depends on the actual DNA sequence flanking the gene…unfortunately you didnt provide the sequence so ill help you out the best i can:
if you take a look at the top strand of the DNA (in the 5′ -> 3′) direction you can design your primers by following a simple little trick. you primer for the 5′ end is the EXACT SAME SEQUENCE . your primer for the 3′ end is the complimentary sequence.

example: 5′ AGGCTTGAGGCTAGACAG——————-GENE——————GATAGCTCTCTGAGT 3′
so for my 5′ primer I would choose 5′-TGAG-3′ or i could even choose 5′-GCTT-3′
normally when you design primers you want a Tm at or above about 50 C, and an approx. equal amount of G/C and A/T
for my 3′ primer i could choose 3′-ATCG-5′ BUT remember to report it in the correct 5′ ->3′ direction, therefore my 3′ primer would be 5′-GCTA-3′. Instead i could choose 5′-TCAG-3′ as my 3′ primer. there are many different correct answers

I hope this has helped a little bit
Also, a 4 nucleotide long primer would be very non-specific meaning that it could bind almost ANYWHERE in the genome, which means you may not amplify the desired gene. primers are usually ~20-25 base pairs in length

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