I took 20 ul broth culture bacteria and added to 180 ml miliqi water. And boiled 5 minutes, followed by keep in -80oC for 10 minute.To got aliquot of DNA, I centrifuge its for 5 minute. I only use 1 ul DNA aliquot for PCR. The amplification was succeed, but only could got 1000 bp fragment. Anyones have suggestion of it. My culture age is 3 days.
It could be the PCR conditions. How long was the elongation time for your PCR? Elongation should be 60 seconds per 1000 bp. If the elongation temperature is too low, the products can be short because of reduced Taq efficiency. There are other potential causes, but without more information about your experimental methods, better answers are not possible.
Dear Paul my initial 94oC for 5 minutes; denaturating 94oC 45 seconds; Anneling 55oC for 45 seconds; elongation for 72oC for 2 minutes. Final elongation 72oC for 10 minutes. I doing 33 cyles. That my pcr condition thank for your input
Dear Agusman, you will try this PCR condition then may be will get 1.5 kb product size.
initial 95o for 5 min, then 30 cycles of denaturating 94o for 30 sec, anneling 57 for 40 sec, elongation 72 for 1.30 min and final elongation 72o for 10 min.
Another method, In PCR master mix, add 10X Taq buffer and MgCl2 separately.
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