Hi, and your DNA (or RNA) extraction worked properly? Which method did you use? Cheers, Nadine

Maybe you can check the Extag or dNTP? In this case, we usually set a temperture gradient from 50oc to 60oc at the same time. Or maybe you can ask another guy to do it for you to avoid some faults you were not aware of when doing the experiment.

I am sorry for the lengthy explaination. But I believe that there are the parameters you should check to make sure.

Ingredients:

1. Primers - well, are you adding enough primers? It is recommended that you should have 0.5uM of Primer. However, in some scenario, 0.1uM would be sufficient for you PCR.Below is a website in which you can submit your oligo to be analyze.Make sure you check for heterodimers, hairpin and so on.

http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/

2. dNTP - this ingredient goes bad pretty quickly. It is highly recommended that you aliquot dNTP just enough for 10 or so reactions and kept most of them from freeze thaw process which degrades dNTP.

3. Are there any chemical inhibitors. Many people use some form of TE buffer to resuspend, elute DNA. However, there is a chemical in TE buffer - EDTA which is a chelator of Mg++.

4. Template - is subject of your PCR pure? You might have to process your template before carrying out cloning. You could also find out the purity of your template by obtaining OD 260/280. A value of 1.8 or higher is more desirable.

5. Enzyme - yes, enzyme decays sometimes faster than the expected Expiration Date. It would be a great idea to obtain another enzyme from someone else.

6.MgCl should be around 1mM to 2mM. Having excess magnesium might produce a smear or additional undesirable bands.

7. Nuclease Free water for your PCR - Sometimes, nuclease free water could be contaminated( no longer nuclease free.)

Protocols problem:

1. Denaturation step: is your DNA have enough time to denature? Denaturation time could ranges somewhere from 20 to 30 sec.

2. Anneal time: Kai Yang is right. You could tried gradient for temperature. Also, you could need anywhere from 20-40 second to anneal.

3. Elongation steps: make sure that you allow enough time for elongation of DNA. Depends on your size, the elongation time could be different. Typically, elongation time should be 1 min, add 1 minutes for every 1000bp more.

4. Finishing Elongation - make sure you also allow enough time for it. Typically, one should allow 10-15 minutes for any partial product to form.

Good luck.

There can be a lot of reasons. If your polymerase tolerates its, you can try to add DMSO (% depending on the polymerase, see data sheet). It has worked so many times for me. You can also try to make a gradient pcr and determine the optimal annealing temperature.

Good luck!

Might be a stupid question but did you try to change other parameters like your electrophoresis buffer, a higher gel percentage, voltage etc?

Hi Erisa, we are still waiting for your answer(s). Cheers, Nadine

Hello and sorry for the delay.. However I am doing a standart cloning method for almost 28 mutants, and the parameters which I have been using is the same for all of them, but it looks that all of them have worked fine except this one. I kind of putting aside this one and moving on to the others, because I haven't tried adding DMSO yet

I actually used another template for this one (mutant#35) and it still didn't work but the point is that I still see bands but not at the right place, and I might be wrong but I don't think is gel electrophoresis which is affecting it, I think is either PCR or the primers... because even though I checked the sequencing ,,,,I don't know.. I am stuck