I'm interesting to know that,

1. how many additional nucleotides can you add at the 5' site of the primer ?

2. How many restriction sequences can you add at the 5' site of the primer ?

3. when calculating annealing temperature is that enough to consider perfectly annealing primer without considering anchored nucleotides?

  • If I design a primer with 20 nucleotide complement to the DNA which it supposed to be annealed perfectly together with additional 20 nucleotides with few restriction sites or any oligo sequences, what are the things to be considered, in primer designing, PCR and restriction digestion?

Thank you :D

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