Yin Chin,

Some of my thoughts are below:

1. Do you have your original DNA sample that worked for you? If so, always include the original DNA with the other samples for the same PCR run, that should tell you if its the PCR or the sample quality. If you don't see the KO bands with the original tube of DNA, then you need to find a good positive control.

2. Have you tested a gradient PCR to find out the optimal annealing temperature for your primers, 65C may be the best for WT but may not be for KO. I would run a gradient PCR for KO from 55-65C and see if any of the temperature gives you a band.

3. The fact that you see WT band in heterozygous mice but not knockout mice suggests that your PCR works with your WT primer and you have good amount of DNA. So it suggests to me that it's the PCR program or KO primer that did not work for your KO reaction. Refer to the specific Taq polymerase you are using, and you may need longer denaturation time to 3min, and shorter time for annealing to 30s, and more cycles to 35 cycles (I have gone up to 38 cycles which gives me very strong band).

4. The cloud with super small size indicates the primer dimer and you don't see it in the WT column suggesting your KO primers did not bind to DNA template and since you did not change sequence of the primer and get same results even with newly bought primer, it is very likely your PCR reaction is the problem.

Hope this is helpful,

Zhixin

try to reduce the amount of DNA, 50 t0 100 ng may be too much. Try to use 25 ng DNA per reaction mixture if your reaction mixture is 25 ul. if your reaction mixture is 10ul, use 10ng DNA per reaction mixture. Sometime too much DNA can create problem.