I'm going to do overlap extension PCR. The final PCR product is composed of three PCR products. The length of each PCR product is 55bp, 850bp and 600bp. The length of each overlap primer is 35bp. Those ends of primer are G or C. The independent PCR products can be amplified well using those primers. Then, PCR products can be amlifed well using the first PCR product (55bp) and the second PCR product (850bp). However, when I did PCR using the combined one and the third PCR product (600bp) or using the second one(850bp) and the third one(600bp), PCR was not amplified. The same phenomenon happened when I did it with other plasmids. Therefore, I don't think those primers are wrong. Does somebody have amy ideas to solve this problem?
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overlapping extention PCR problemoverlapping PCR
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