Dear Oadi,

2 ng/ul DNA is generally a low quantity. However, the concentration of the DNA needed depends mainly on its lenght. For example if your PCR fragment is 100-300 bp long, 2 ng/ul could be OK. Keep in mind that every 400-600 bp of lenght you have to add 2 ng/ul more (for example 4-5 ng/gl for 500-800 bp, 6-8 ng/ul for 1000-1500, 8-10 ng for 1500-2500 bp).

Concerning the noise, it can be due to different factors.

First, it is possible, especially if the PCR product is long, that you have an aspecific annealing of your primer, i.e. it anneals in two positions, so you have two signals. In this case you should design a novel primer. Secondly, it is possible that your PCR product is "contaminated" with your amplification primers, so the final signal is given by two independent signals which are superimposed. In this case it is difficult to eliminate the residual primers since although most kits eliminate most of the primers, a small quantity of the them remains in the PCR solution. Keep in mind that if your PCR produces a low quantity of DNA, you have to add in your sequencing reaction a greater volume of PCR solution and thus a greater quantity of your PCR primers remained, In this case you should increase your PCR yield in order to reduce the volume added to the sequencing reaction, for example adjusting the Mg2+ concentration, the template concentration, the annelaing temperature or the extension time.

Best,

Enrico

A low DNA quantity (like you have) can cause overlapping peaks, which gives poor quality sequences. But again, it also depends on the length of the sequence, longer sequences require more DNA. Also, the purification step after your PCR run affects the quality of the sequence. Finally, it is very important to make sure that your primers aren't annealing to multiple locations on the sequence & that there are no trace PCR salts present when sequencing, because these can affect the sequence quality as well.

Thanks for Enrico and Naji for you clearly, may be my question was general, I do the sequencing for 700bp length fragment and i use concentration 2 ng/ul of PCR product after cleaned by using PCR product clean kit.

So may be the low concentration of DNA is the problem?

Tomorrow i will upload the sequencing file, so it will make more sense.

Please find the attach file for the sequencing that I got for TEF gene.

I think there are two issues in your sequencing. In both cases the signal decays too early, around 100-110 bp in one and 250-280 bp in the other. This can be due to a low template concentration. In the second sample, in addition, there is a lot of noise, and this can be due to an aspecific annealing of your primer. If the template is the same, and in one sample there is noise whereas in the other there is not, it is most likely the the noise is due to an aspecific annealing rather then to the presence of residual primers. (In that case you would have been observed noise in both samples). Thus my suggestion is to increase your template concentration and to change the first primer you use to sequence.

I will suggest to use at least 70-100 ng of total PCR product in sequencing reaction and the total amount of sequencing primer should be between 3-5 pico moles per reaction. Ratio of primer and template is very crucial for success of sequencing. All other suggestions from Enrico and Naji are worth considering. One more thing you may want to make sure your sequencing primer is not degraded. 

Good luck

Dear Oadi

I want to support the recommendations made by Enrico and Naji to increase the pcr product for sequencing a 700bp amplicon. I could not see your sequences (don't have software for the extension .rar), but according with descriptions made by Enrico I have a metal picture of your results.

Here I want to add some information: You haven't described the pcr cleaning procedure you applied. If you used column based cleaning kits (e.g. Qiagen purification kits), be aware that you are losing a lot of pcr product that is washed away. I would like to recommend you to clean with enzymes. They are very reliably, handy and cheap (much cheaper than columns). You will find ready to use products (Illustra ExoStar from GE) or you mix the enzymes by yourself (exonuclease I and Fast-alkiline-phosphatase from Fermentas), what is even cheaper (cf. file of products).

Please, consider this point since you don't have NO loss of pcr product if cleaning with enzymes. You can load your pcr products on a agarose-gel and if they fullfill all conditions for sequencing (single band, enough concentration), then clean it with enzymes and DIRECTLY perform a cycle sequencing reaction. You only need the enzymes, pipets+tips and a pcr machine.

good luck!

carolina