PCR Optimization for 50-100 base transcript - How do I increase yield of small PCR product? (Oct/03/2005 )
Hello,
I'm trying to PCR 50 and 100 base size transcript. With my standard PCR protocol, I get the right size for both of my products, but the yield is very low. My protocol includes adding in 2ul of DNA template at 0.5ng/ul, 2ul each of both primers at 25uM, 2.5ul of Taq polymerase, 10x KCL buffer w/o MgCl2, 10ul, and 10mM dNTP mix at 2ul, and 2ul of 25mM MgCl2 (total reaction volume is brought up to 100ul with H2O).
My typical yield is about 2ug. How do I increase my yield w/o comprimising the quality?
Thank You.
I am confused by your post, are you trying in vitro transcription or regular PCR or RT-PCR? Please elaborate.
I'm doing just regular PCR, with a denaturing step, additional denaturing for 30seconds, annealing, and extension step at 35cycles, and at the end of 35 cycles, do an additional extension for 15minutes and then 4 degrees hold in thermocylcler.
Dear Nanopower,
I think the low yield is due to tow factors:
1) low amount of starting material. You might thinking of increasing it. I usually use 20-50ng/ul.
2) You MgCl2 is too low. 2ul of 25mM MgCl2 in total 100ul is 0.5mM. increase at least up to 1.5mM.
Best regards
I'm trying to PCR 50 and 100 base size transcript. With my standard PCR protocol, I get the right size for both of my products, but the yield is very low. My protocol includes adding in 2ul of DNA template at 0.5ng/ul, 2ul each of both primers at 25uM, 2.5ul of Taq polymerase, 10x KCL buffer w/o MgCl2, 10ul, and 10mM dNTP mix at 2ul, and 2ul of 25mM MgCl2 (total reaction volume is brought up to 100ul with H2O).
My typical yield is about 2ug. How do I increase my yield w/o comprimising the quality?
Thank You.
2ug of such a small fragment is pretty good considering that you have no free Mg in the reaction. You must be adding more than you say - maybe check the recipe or check that your Mg is really only 25mM.