Hi, I an joining three fragments - 312bp, 937bp and 313 bp by overlap extension PCR. What I get is bright smears all through the lane, starting from the well down the lane. While the longest fragment I can form is only 1.5kb, why am I getting smears that start well at the beginning from the well and runs down the lane downwards?
Reaction conditions -
Individual fragments gel-purified after amplification (260/280 value is >2 for all of them)
OEP -
step 1-equimolar amounts of each of the fragents, 200ng total 25ul, tried with 400ng, tried from 20x to 60x cycles
step 2
end primers + buffer + dNTPs + pfu = 25 ul addition to the step 1 mixture - 30x cycles
Try combining all three pieces in lower concentrations, like 10-30 ng per piece in 100 uL total volume. The molarity of the fragments needs to be the same, not the mass, since they are all different sizes - 30 ng of P1 and 3 with 10 ng of P2. Also combine the end primers and do the PCR in 1 step instead of two. That has worked for me to generate genes up to 5 kb with only one main product.
there may be problem during DNA extraction problem. Perhaps it may not be intact. Try to evaluate the quality of genomic DNA u have extracted on agrose gel. If got intact band then you can go for above mentioned mentioned suggestions given by various experts.
Thanx Matthew, Latif, Rita, Ajay, Iskander for your valuable suggestions.
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