The signal was high at the beginning and then decreases rapidly. This is because the concentration of the template is too strong. you have to dilute your sample good luck.

Almost certainly we have an empty vector, and the signal you obtain refers to the vector itself. My suggestion is to check the presence of you insert by digestion and gel visualization.

I think you are using too much of plasmid or primer, or both. All your labeled nucleotides are used up. Here is a good resource that describes lots of the problems that are encountered during sequencing:

http://tools.invitrogen.com/content/sfs/manuals/cms_041003.pdf

Chapter 8 is the troubleshooting chapter that discusses your issue.

Have you check the inserts with restriction analysis prior to sequencing? Probably there was contaminations.

I don't know how much plasmid DNA are you actually putting into your sequencing reaction. Also I have no idea of how big your final plasmid construct is? Say if the size of plasmid you are trying to sequence is 6 kb, you will need 400-500 ng of plasmid and 3-4 pico moles of primer in one sequencing reaction.

The signal was high at the beginning and then decreases rapidly. This is because the concentration of the template is too strong. you have to dilute your sample good luck.

In my experience, this problem sometimes occurs because of a disequilibrium between template and primer concentration in the sequencing reaction. I suggest to revise both concentrations and adjust. Good luck!

Hi Reeteka,

this typically sound like a secondary structure which may arise from your insert. During seqeuncing reaction the insert forms a hairpin loop (partly double stranded), which means the polymerase drop off suddenly. Sequences which are often affected by secondary structures are mostly GC rich but also for GT rich inserts these features are known. If you have a GT rich sequence region it helps to sequence the insert from the reverse site (this does not help for GC rich regions which form sec. structures on both strands). For GC sec. structures which are very strong and immune against heat denaturing (during seq. reaction) you can linearize the plasmid with a unique cut in the insert (preferably within the loop).

In general there are some additives to add in every sequencing reaction to stabilize the denatured strands and prevent more or less successful back folding (these additives are usually used by sequencing companies).

hope this helps

GOOD LUCK !

Anke

Hi,

a bit more info on the experiments would probably help the troubleshooting.

What is "multiple inserts"? One construct with many fragments ligated together? or many independent cloning of single inserts at a time?

In the latter case what is "sequencing primers ... going into the inserts"? Do teh 5' end of the primers align with the inserts? Do you have similar inserts or you have primer pairs for each insert?

Is it a capillary sequencer and dye terminator chemistry? Then the "around the time" that the signal should emerge may be the front when all the unincorporated dye and monomer and all that junk comes through. IF nothing comes after, then you may not have working sequencing reaction at all, which suggests no reaction at all, so that no annealing or some missing reagents, rather than suboptimal primer/template concentration.

Have you checked by conventional PCR whether the sequencing primers anneal with the constructs?

If you can prove yourself that the right inserts are in the plasmid (by agarose gel assessment of the right size of restriction fragments of the constructs and right size of PCR amplicons) THEN you should go for less trivial solutions.

If you have the right concentration of templates and primers, you may try linearizing the plasmid at a unique restriction site on the plasmid somewhere far from the insert, if any such site exists.

Hi!

As others already suggested you, you should try to digest your minipreps before sending them for sequencing to make sure you have actually cloned the inserts into the pcDNA vector. I don't think the problems you're facing are due to the DNA and/or primers concentration...

Good luck!

Alessia

Confirm your insert is cloned by PCR using primers specific for plasmid regions that flank your insert. Only after you make this confirmation you can figure out whether your sequencing template and primer concentration are the problem. Good luck!!

That's interesting as I had the same problem. Even more confusing, the sequence from the forward primer was OK and went into the insert, but the sequence from the reverse primer just dropped off exactly at the restriction site I used for cloning. It wasn't the sequencing, as I got 300 bp of great quality before it suddenly stopped and I also used a primer within my insert for control - this primer gave me great sequence of about 800 bp. I have no idea what causes this - running plasmid on the gel, performing restriction digest with a dicutter that cuts in the plasmid and in the insert all looks as it should. It seems like there is something on the plasmid in that spot where the insert was ligated. In the end now I am repeating the cloning with a different restriction site. If it's not too much hassle for you, maybe you could just start from scratch?

Again, Anna's story confirmed me in the suggestion of a secondary structure. she also told that the results with forward primer were ok. As I mentioned above, I would first recommend sequencing from the other site.

It is not clear how much primer or template concentration you are using. You give the concentrations but not the volumes used in your reactions. 100-300 ng of plasmid template and 3.2 to 5 pmole of primer in 10 ul sequencing reaction is appropriate. Assuming everything is OK with your instrument

1. First, make sure that you have good quality template

2. Second, confirm by PCR the presence of your insert using two sets of primers that will generate different size fragments on gel (example: two different forward primers away from the insert use with the same reverse primer within the insert).

3. Once confirmed, performed sequencing using preferably the two forward primers above or different but similarly-spaced primers at concentrations adjusted to the manufacturer’s specifications. This will allow you to troubleshoot for any problems associated with secondary structures.

4. Use the same template prep for both PCR and sequencing

5. Good luck

Thank you all for helpful suggestions!

From the latest attempt- it does seem that secondary structure is contributing to the problem - when sequenced using a protocol for 'hairpin structure', we did get good quality sequence from the complementary strand.

This could be related to diproportionate concentration of primer or template. Also, you might consider if it could be a primer-dimer peak.

I agree with Maged. In your sample the Template quantity is very high, because you know in Cycle sequencing process your intial template is being used every time as a template.

I use generally template beween 100-200 ng total template and 5-6picomoles of primers. when you use higher concentration of template all the raw material will be finished in the amplification of only few nucleotides of your template.

Agree with Peter's comment. Seq. reaction is not working.

Also agree, use less template. You can even go lower as well with your primer. Good luck!

The peaks that you see at the beginning of the sequence file are probably unincorporated dye going through the sequencer, these are usually around the 70 bp mark after your primer. The drop off could be down to a couple of thing, too much salt in the reaction will do this. But by the sounds of it the sudden drop off, might be caused by palindromic sequence around your cloning site. Have a look at your expected sequence; if you have cloned MCS’s back to back then these will form hairpin structures in the sequencing reaction. So look for something like this: BamHI, HindIII, NotI, SacI, NotI, HindIII, BamHI. The reaction fails at or around the BamHI sites in either direction. I hope this helps to explain it, but if it is this, you will never sequence completely through it.

Thanks Mark, especially for the point you made about MCS composition. Good point to remember! I am using pCDNA3 vector for this cloning (attached file showing MCS), which goes like this:

HindIII KpnI BamHI BstXI EcoRI EcoRV BstXI NotI XhoI XbaI ApaI

I did a double digest with EcoRI+XhoI to linearize

After ligation, and before transforming bacteria, did a final NotI digest, which would linearize the original plasmid, but has no site in the new (correct) plasmid.

In reference to your point, it doesn't seem there's back to back MCS. But that said, running a "hairpin protocol" on the sequencer did help get better output.

Please have a look..and let me know your opinion- should we use a different vector?

Thank you!

Hi Reeteka,

I don’t think it is a problem with the vector, I would check you insert for hairpin like structures around where the sequence drops off. Presumably you are using T7 and Sp6 primers to sequence. Could you design a couple of primers coming out from your insert? You may have better luck sequencing in the other direction. Also, add an extra 0.5 ul of Bigdye to the reaction (10ul reaction) sometimes helps, you can blast your way through with difficult samples. I hope this helps and good luck.

Thanks so much, Mark! That's very helpful to know.