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Annealing temp selection

Annealing temp selection - DNA Forum

Annealing temp selection - Discuss DNA, the molecule of hereditary. Topics include DNA structure, DNA replication, DNA function.


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  #1  
Old 05-26-2010, 09:44 PM
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Default Annealing temp selection



I am using the following primers for a PCR

5'-CAC TCG TCC GAG GGC AAA GGA ATA-3'
Tm 54.1

5'-TCA CCC CAT TAT CAG TTT TGT AGG-3'
Tm 60.7

Product is 1140 bps

I'm using Invitrogen's Platinum PCR SuperMix:

1 ul each primer
12 ul supermix
2 ul template

I am wondering how to calculate the best annealing temp. In the past we have just used a touchdown program but it is long, and I'm wondering if I get it faster entering my own program. I've done gradients before to determine annealing temps, but I don't want to waste supermix right now because our company is on the thin edge $-wise. Suggestions?

We do DNA extractions with 1X TE

TIA
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Old 06-01-2010, 04:45 PM
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Default Re: Annealing temp selection

As they say: fast, cheap, and good, pick 2. If you try to do it cheap and fast, you're not very likely to get good results. If you want to get good results on the cheap, your best bet is to stick with the longer touchdown protocol to avoid having to optimize the annealing temperature.
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Old 06-02-2010, 04:23 PM
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Default Re: Annealing temp selection

Agreed with bonnie, but if you really wanna pick an annealing temperature, I'd say 55-56 C. Good luck!
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