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I am trying to do a intergon pcr... The pcr machine I am using has this "asterisk" which indicates for the "ramp" function. I am using 68 degrees celcius for my elongation temperature.. and 4 min elongation time.. does the ramp also increases temperature after every cycle? I only know the elongation time is increased every cycle. is this optimised temperature? should i change it to 72? somehow someone has been using 68 degrees and thus i am hesistant to change the temperature as i do not know if it may be optimised. what should i do?
I am trying to do sequencing on the entire gene expecting about 4kb, thus i was advised to use 4 min elongation time.. i want to know when i get my pcr product on a gel, would it shows up around 4kb size? or roughly 1000 to 1500 bp?
Sorry if i sound confusing.
I must admit I am a really lousy in this! Sorry.. =(
Thanks
