Error-Prone PCR for Mutation Library Generation

[jiajia1987]

Dear all,

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!!!

[xinlong]

First, I want to ask you: do you have experience of good work on it? If you have, you can try to repeat that work in advace and see if it works well again.
You can ask your partner to do the same work(even the ligated product should be same ), and see if she can get the good results again.

[jiajia1987]

Dear xinlong,

This project is new and I am the one who is starting on it. I have tried many ways to get the library but it doesn't work -- I still end up with a smear whenever I am at that particular step while my partner does not. It works perfectly fine for my partner and she uses the exact same reagent as me. Even my supervisor is really puzzled as to why it is not working when it should. I am unable to get her to use my ligated product and do it for me as she is busy with her own project.. sigh. I am trying out another method for these few days to see if it will work. Hopefully, I will get the library I want.

[jiajia1987]

Dear xinlong,

This project is new and I am the one who is starting on it. I have tried many ways to get the library but it doesn't work -- I still end up with a smear whenever I am at that particular step while my partner does not. It works perfectly fine for my partner and she uses the exact same reagent as me. Even my supervisor is really puzzled as to why it is not working when it should. I am unable to get her to use my ligated product and do it for me as she is busy with her own project.. sigh. I am trying out another method for these few days to see if it will work. Hopefully, I will get the library I want.

[xinlong]

I never suspect your operation on PCR. So, I wont give your some suggestion on operation, though some error operation can lead to smear bands. I think your ligated product is not good enough Because the only difference between you and your partner is the ligated product. On the other hand, you can do a negative control to see if there are smear bands in the negative control.
Good luck.

[jiajia1987]

The negative control does not show a smear band, so I suppose the problem lies with the ligated product. I am trying to see what is wrong with the ligated product. But nevertheless, thanks a great deal for the discussion!