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
In general practice, 5' ends of primers are flanked by a single restriction enzyme. We add 4-5 GC rich bases at 5' to that, in order to enhance annealing to the target sequence. Theoritically speaking, annealing temperatures calculated without the additional bases work just the fine, but in some circumstances you might have to raise the Ta to attain a specific band.
I would like to learn from you the advantage and purpose of adding multiple restriction sites to your primer.
Thank you Ms. Rituparna.
Yes so far I also did the same way. But Im planning to construct a vector in my future experiment. So if I could use Atleast two restriction sites per primer it would easy for me. I could just bypass few additional steps.
I woukd like to know anybody who try it earlier!
Thanks
Primers can be as long as you like (or as long as your provider can synthesize i.e. ~100 bases long), so you can add as many R/E sites as you like on the 5' end. TM you count only the nucleotides that anneal to the target template (i.e. 60C) for the first 5 cycles and then you can move to treating the product as template and if the primer is really long then a 2-step PCR (94-72) for the next 25 cycles is best.
Do however consider that primer synthesis is full of mistakes and the longer the primers the more clones you have to sequence over the annealing junctions to find a good clone.
Good luck
Thanks Yoel,
Your words were really helped to clarify lot!
Still I have a doubt of the percentage of anchored nucleotides! I mean, 20-25 additional bases (x) to 20 bases that anneal to target (y) (~50% extra nucleotides in the primer), will it be Ok? So totally the primer (x+y) would be 40-45 bp.
From your answer I feel I can do it, but could you please advise me the perfect length of x and y I can use to design the primer?
Thanks again!
Hello Nilmini akka,
I hope you are doing Gibson assembly to generate the vector. You can add many restriction sites as you like by keeping them in the overlap.
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