no band after long-distance PCR - (Feb/22/2006 )

Dear all..

i did my pcr to ampify 9 kb of my genes. Unfortunately i got no band at all for my samples (my samples are putative plasmid that i got after ligation),but there was a good sharp band for the positive control that i did on the same PCR condition.

The question remained here was how on earth i cant get my desired band for my samples but there was a good band for the positive control then?

For your information,the positive control was the same backgroud vector for my samples.And i designed the primer according to the origin vector.That the same for both of them.


my pcr condition

initial denaturation : 94 deg,5 min
subsequent step : 94 deg, 1 min
annealing temp : 55 deg,30 sec
extension temp : 72 deg,9 min,30 sec
repeat step 2 30cycles
final additional : 75 deg,10 min
hold : 4 deg

for your kindness and attention,i thank you.

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How big is the band you get in your positive control? The longer any fragment, the more diffficult for any enzyme to amplify.

Apart from this: which enzyme are you working with? What's the amount of starting template (and the quality of your DNA)?

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for the positive control i got 5 kb band.i used takara extaq polymerase in this PCR..the starting template was < 500ng ..

but i did repeat my PCR yesterday,unfortunately i got no band (even for my positive control! )..
i increased the dNTP and extension time..

why...this happened to me?

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Did you add a proofreading enzyme in there, also try increasing the cycles for a 9KB product 35 cycles isnt enough

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