I am trying to clone mouse Mrp4 (started from cDNA), after designing four primers (4-1R,F,,4-2 F,R,, 4-3 F,R,, 4-4 F,R) with overlap more than 20 bases, I got four segments around 1000bp(expected length) with overlapping parts ( I used KOD plus ver 2 kit).
after that, I tried a 2nd PCR for these fragments using primer 4-1 F and primer 4-4 R and the PCR reaction was :
3steps cycle
94c 2min
98c 10sec
(50,55,58,63 tried different TM at a time)c 30sec
68c 4:30 and then later I tried for 5min
I am supposed to get a band around 4000bp but I'm getting one near 300bp
please, someone, help me I'm desperate!
Hey Ella,
I see that you already tried gradient PCR. What is the Tm of your primers?
You might try a touchdown PCR by setting a higher annealing temperature at the first cycles and incrementally decrease it in the following ones. That should help you to increase the specificity of the reaction.
The other things that you might try is a lower template concentration (higher concentrations tend to decrease the amplicon specificity). I would also suggest decreasing the temperature for denaturation unless required due to high GC content.
what is the overlap of the amplimer?s. If 20bp as you say then this may be too short and the amplimers will just re anneal with itself and no longer fragment will be produced to amplify further. You could try the second amplification in 3 steps so melt and reanneal 1f-1r and 2f-2r together then amplify the 2kb fragment 1f-2r. Then melt and reanneal 1f-2r product and 3f-3r product and re amplify 1f-3r for a 3kb fragment and repeat for 4f-4r for the full length amplimer.
I assume you have tried the single amplification of your cDNA 1f-4r directly.
I think that having primers generating a much longer overlap would probably give you a much better chance of success as this would generate much more of the longer product at the annealing stage. Possibly you would only need to redesign 2f 3f and 4f primers
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