This is my first time over here in this forum and I would like to take the opportunity to get something sorted out.
I am trying to make a mutated library for selection. Basically, what I do is to do an error-prone PCR, followed by Dpn1 digestion and then an amplification to amplify the mutated PCR products left behind. This PCR products are cloned into a vector for transformation so sequencing can be carried out to characterize the library. The ligated products are also subjected to an amplification using biotinylated primers because I want a biotinylated mutated library.
Now, the problem is that though I do get occasional problems here and there for the PCR steps, they are often solved via different means. The step that is giving me a lot of problem is the PCR step using biotinylated primers. Whenever I do this particular PCR, I always get a smear in my gel results and I don't know why. I have tried diluting the template (which is the ligated products), changing the water, increasing the extension time, increasing the number of PCR cycles but none works. My partner in the same lab uses the same reagents as me for this particular PCR too and the only thing that differs between us is the ligated products, which is also our template and it works perfectly fine for her.
Do give me some advice as I have been at this for two months and time is running out for me. If this doesnt work soon, I will have to try another way of getting my mutated library but I would want this to work out because of all the work and effort I put into it. Thanks in advance!!!