low intensity signal with peaks under peaks

[mohd2b]

thanks guys

well my template is pol HIV gene

this is kit based methods. I use Viroseq kit to test for resistance. I have used it in my training in Germany and now iam optimizing it here in our lab.

Yesterday we run standard BG v1.1 rxn for sequencing and we had a very nice sequence with high quality using v1.1 analysis and istrument protocol.

iam now runing same samples but using new primer master mix cycle sequencing rxn and load the plate today and wait to say

[sequencingfan]

Do You always set sequencing reactions on chemistry version 1.1?
Did You check the protocol on instrument that is set up for version 1.1 not for 3.1?I think there are different mobility files etc.
Of course it might be a standard problem with double sequence as nucacidhunter mentioned in last post.

[nucacidhunter]

There could be few reasons: 1) more than one template in the sequencing reaction,; 2) non-optimal primer which primes sequencing reaction from more than one location; 3) non-optimal reaction condition. What was the template?

[mohd2b]

thanks for reply sequencingfan

Well after receiving the analysis software we have seen that there are some setting we need to modify in analysis protocol we used.
We managed to get a quite good sequence but still not very good for analysis.

I have checked the sequence for other test carried in the same instrument and i realized that despite having low signal on the raw data they still have better and readable sequence!!??

We are using the AgentCourt clean-up kit after cycle sequencing and it is the same kit the other group using. however they got good results and we not.

my final cycle seq rxn volume is 10ul (6ul primer buffer mix, 2ul PCR product, 2ul water)

Could it be possible that the volume of PCR is two low or two much?

[sequencingfan]

Hello,

in my opinion the average signal strenght for all bases is very low ( below 100 ) and the software pull up the background to maximaze the real signal thats why You get double signal but it's only my opinion.
Do You suspect any inhibiotrs in sequencing reaction?
Meybe the clean up metod is not good?

[mohd2b]

Hello
well iam doing sanger sequence for samples that used for resistance analysis. the bigdye chemistry is v1.1

after getting the sequence trace files raw data has shown low signal intensity and the electropherogram show peaks under peaks or double peaks almost in all the sequence.

the sequence quality value using Sequence analysis software show red color but in some regions blue color.

What could be the reasons?
what is the troubleshooting from experience?

thanks