2. By changing the thermal cycle, it can play a little role. My method is 50-60C magnification 10 times, and the extension time is set to AC and BD; Next, the annealing temperature was directly raised to 72C, 30 times of amplification were performed by two-step method, and the extension time was set as the extension time of AD. Although the ideal results have not been obtained, the most fragments are AC and BD, and the long fragments are very, very weak.
3. As Mybbff moderator said, I also tried to adjust the primer concentration. I have seen Han Jian's patented technology TEM-PCR, in which the key point is to adjust the primer concentration to realize fragment enrichment. Combined with the temperature change, I reduced the concentration of B and C to a very low level, and I achieved a difference of 50 times between A and D, and B and C. My idea is to adjust the concentration of BC to a very low level, so that it will be exhausted before the logarithmic amplification period, and only primers with very high TM are allowed to extend the next 72C.
4. At present, I haven't got the ideal result, and the amount of long fragments is far less than that of AC and BD. My idea is to pay more attention when designing primers and overlapping regions. There must be a big difference in TM value of primers, a, d >；; Primers b, c, a and d are slightly longer. In line with these changes, and then adjust the program, I believe it will be successful.