While using the overlap PCR method for site directed mutagenesis, whenever I'm trying to anneal two fragments (A-1700bp, B-900bp), I'm getting a final product band at 900bp (i.e. only band B is getting amplified). The total size of my product should have been 2600bp.
Can anyone please help me to troubleshoot this problem?
Note: I'm not using any kit or phosphorylated primers/T4 ligase.
My overlapping primers are 40 mers each, with an overlap of 20 bps.