Hi Kimberly,

It might be a little helpful if you describe the junk sequence you are getting in your results. Does it appear that the sequence simply failed? Or are you seeing a polyT region followed by what we often called the sailfish effect. It is common for cDNA to have these polyT regions causing stuttering, basically all 4 bases seen in every position. Have you tried to sequence in both directions or just 1 direction?

I agree with Doug. You need to describe the "junk" you are seeing. Also, have you done control sequences at the same time? This might tell you if there is a reagent or machine problem. Please be more specific about your methods, etc.

Perhaps sounds like a job for PacBio. If you pay just for reagents, it is cheap and 5k bp should be a piece of cake for it - have a look around for a friendly lab with access to this piece of heavyweight equipment :)

Hi all - my control sequence works fine so my reagents and instrument are fine. I have tried sequencing in both directions with a couple of different primers. Sometimes I get no sequence and other times I get a few readable base pairs (no evidence of polyT region) and the rest of the sequence is literary junk - words fail me - peaks of two or three bases, overlapping peaks, etc.

Thanks Kimberly,

It sounds as though you are not getting any amplification. The fact that you have sequenced with both forward and reverse primers would eliminate the possibility of a polyT region. Assuming that the cDNA has been inserted in a plasmid, have you checked the plasmids by agarose electrophoresis? If so, did you see one or two bands. One band could indicate the DNA is nicked.

Any possibility that you may have a mixed population of genome fragments? Trying to sequence multiple different fragments at once can lead to "junk" sequence output.

Hi Kimberly,

Have you sequenced huge streches before and Is your control sequence 5kb in length. I have used the ABI 3730 analyser and BigDye Terminator v3.1 Cycle Sequencing Kit and I could only achieve a stretch of 1.5kb. If your control is smaller in length compared to sample then may be the problem is with the length and may be you should try other methods.

Hi kimberly

I agree with Kevin Diebel. Could you please check the concentration of your template. In case of sanger's sequencing method it is not possible to get sequence of 5kbp length. Alternate methods may be tried.

Hi Kimberly, I recommed that you try to clone the fragment into a vector and try to sequence it using primers derived from the vector.

Hi Kimberly, I agree with Kevin Diebel. You may have a mixed population of fragments. The best solution would be to clone your products (in PCR cloning vector, like TOPO from Invitrogen); and sequence single clones ( although it sounds time-consuming, it may actually be the fastest way)

I absolutely agree with Daria.

I, too, concluded that I may have mixed population even though I gel purified the 5 KB band prior to sequencing. I am currently trying to clone my 5 KB fragments into TOPO XL ad then will sequence individual clones. In respond to Aruna and Arjun, I was not trying to get entire 5 KB in one section rather I have primers every 500 bp or so along length of DNA

I would suggest "nested PCR" design. You basically will do multiple sequencing and them alligne the sequences and make contig. I suggest using Vector NTI for the analysis.

Hi Kimberly,

Cloning the fragment is a good way to sequence. Good luck. Please let us know how it turns out.

I absolutely agreed with Ms. Sharareh Siamakpour-Reihani suggestions. In your case, "nested primers" should be designed and used before multiple sequencing. For cloning, you also can use pGEM-T Vector System.

I haven't enough information to judge, definetely 5kb DNA strech would never come out from Sanger sequencing, as someone said before maximum is 1.5kb.

that's for one. So, you should try with internal primers, do you know a priori the expected sequence you have in there??

cloning would improve results... but first is to know if the sequence in those 5kb is know......

good luch

Hi kimberly,

You say that you gel purified the 5 KB band prior to sequencing. Most probably while you gel purifing your sample, you are exposing DNA sample so much time to UV. It cause primidin dimer and BigDye sequencing failing. Try as posible as short time to UV and try sequencing again. I think this solves the problem, and you successfully sequence the PCR product not need to clonning.

good luck

Hi Kimberly,

we have tried to sequence long fragments, and lowering primer concentration helped. how did you purified the fragment from agarose gel? we made some changes in the column-based method to be sure that the purified fragment had no ethanol at all, centrifuging more time the columns... It is difficult to think that you have many different fragments in the same band... but if this is the problem, you should find the solution with the cloning you are already trying. Let us know ! good luck !

Well, as someone commented before, 5Kb DNA fragment is too long for Sange Sequencing due to the most affordable size for Sanger Sequencing is 1kb. So my recommendation are 2. The first one is to cut the fragment by Restriction enzyme (which produce sticky ends) to produce small fragments of the desired 1kb and clone the fragments in a vector, and sequence them using primers designed for the vector, and alternative is sonicated, run a gel, cut fragments of about 1kb and clone & sequence. Or design a primer walking experiment (if you know the sequence, that could be not the case).

I hope it helps you.