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Sequencing trouble - (Oct/25/2011 )

Hi guys,

i got some problems with sequencing recently.

i just assembled expression cassette (~3.1kb) with overlap extension PCR, and cloned into a expression vector. Then i used outer primer pair to PCR out the fragment of interest. Although the primers are kinda long (~40bp) because they were designed for OE PCR, I got a quite clean band of interest. So i sent the cloning vector with the primers out for sequencing. but failed. Tell from the trace of sequencing results, whole sequence is non-specific from the start of the sequence. i asked for the help from the company. their support told me maybe my primers were not good. so i cloned the fragment into Zero Blunt TOPO cloning vector (invitrogen) and using colony PCR and enzymatic digestion verified the clones. when i got the positive clones, i isolate the plasmids and used T3,T7, M13 to PCR out the fragment. i did get my band of interst. however, still there are some apparent non-specific bands in PCR products. So any idea what's going on? does this kind of sample work for the sequencing company? By the way, my target fragment is AT-rich (62%). do you think that has negative effect on sequencing?

I have to admit that i don't know much about genetic engineering stuff. so any of your suggestions would help. thanks.

Leo

-doorsea-

First of all, most decent sequencing facilities should be more willing to troubleshoot with you instead of just telling you to get new primers.

As for your sequencing issues, its difficult to tell what happened, but any time a template is specifically enriched for one nucleotide or has low complexity regions, sequencing becomes more difficult. Most sequencing cores have alternative protocols for AT or GC rich templates, which can include different cycling temperatures and/or nucleotide concentrations. I would tell the sequencing facility this when you send in the sample and see if they can tweak their protocol to get you better results.

Are you trying to sequence plasmid or PCR product? Sequencing plasmid tends to give better results than PCR product, so would go back to sequencing your plasmid if you can. Other than that, it's difficult to troubleshoot without the chromatograms. If you can post or link them, we can take a look and possibly give better suggestions.

Best of Luck.

-allynspear-

allynspear on Wed Oct 26 13:26:13 2011 said:


First of all, most decent sequencing facilities should be more willing to troubleshoot with you instead of just telling you to get new primers.

As for your sequencing issues, its difficult to tell what happened, but any time a template is specifically enriched for one nucleotide or has low complexity regions, sequencing becomes more difficult. Most sequencing cores have alternative protocols for AT or GC rich templates, which can include different cycling temperatures and/or nucleotide concentrations. I would tell the sequencing facility this when you send in the sample and see if they can tweak their protocol to get you better results.

Are you trying to sequence plasmid or PCR product? Sequencing plasmid tends to give better results than PCR product, so would go back to sequencing your plasmid if you can. Other than that, it's difficult to troubleshoot without the chromatograms. If you can post or link them, we can take a look and possibly give better suggestions.

Best of Luck.


Hi allynspear, thanks for your prompt reply. i just received the 3rd failed sequencing results. The following is my situations:

1) First time, I used PCR primers as sequencing primers and got failed sequencing results.
I assembled expression cassette (~3.1kb, 2.1kb) with overlap extension PCR, and cloned into a expression vector. Then i used outer primers pair to PCR out the fragment of interest. Although the primers are kinda long (~40bp), I still got the quite clean bands of interest (fig1). Even so I gel purified the pcr products and sent them out for sequencing with same PCR primers but got failed results. So I asked for troubleshooting assistant from technical support. The following is q & a

Q: My samples were from gel purified PCR products which were PCR out with constructed vectors as templates. Do you think my samples contained different fragments with almost same size? If that so, i need to optimize the PCR condition. Is it necessary to ligate my pcr products into cloning vectors before sequencing? On the other hand, my primers pre-mixed with samples are kind of longer (~40 bp), which are purified by PAGE. Do you think this have the negative effects on sequencing? Hope you can give me your valuable suggestion! Thanks
A: Based on the information you have provided, and assuming you have ruled out the primer binding to more than one location, it is likely that there is another fragment of a very similar size. In order to reduce the chance non-specific binding, we often recommend sequencing with a primer that was not used in the original amplification. This might require the use of internal primers to attain the full sequence (depending on product size), but it helps reduce the chance of the unrelated product being sequenced. However, if your goal is to isolate the DNA template that you are sequencing then cloning the products into a vector will more useful. Because your reaction is working with the original primers, it does not appear that the nature of their creation is playing a role.


Fig 1

2) Second time. For the convenience, I sent my expression vector and primers used in OE PCR for sequencing. Failed again.

3) Third time. it seems I have no other options. Therefore, I cloned the gel purified pcr product into Zero Blunt TOPO cloning vector (invitrogen) and using colony PCR and enzymatic digestion verified the clones. when i got the positive clones, I isolated the plasmids and used T3,T7, M13 to PCR out the fragment. I did get my band of interst (1,2,3,4). However, still there are some apparent non-specific bands in PCR products(fig 2). I wanna try my luck just sent out my samples. Failed again!

Fig 2

So do you have some ideas about what’s going on?


leo

-doorsea-

by the way, i uploaded the trace files. please check it out. it seems my samples were contaminated. however, i cloned pcr products into vector and used universal primers for sequencing. i cannot figure it out.
Attached Image

-doorsea-

The trace file is pretty difficult to read at the size you have uploaded, but from what I can see it looks as if you have good sequence. Are you certain there is a problem? What makes you think you have bad sequence? Can we look at the .ab1 file?

-phage434-

I agree with phage434 that the sequence in its small format is difficult to read, but it looks okay. Is it just that the sequence is not what you expected?

Just so that I am clear, did you send out the plasmids for sequencing this last time, or your PCR products?

-allynspear-

allynspear on Thu Oct 27 14:00:33 2011 said:


I agree with phage434 that the sequence in its small format is difficult to read, but it looks okay. Is it just that the sequence is not what you expected?

Just so that I am clear, did you send out the plasmids for sequencing this last time, or your PCR products?

phage434 on Thu Oct 27 13:03:19 2011 said:


The trace file is pretty difficult to read at the size you have uploaded, but from what I can see it looks as if you have good sequence. Are you certain there is a problem? What makes you think you have bad sequence? Can we look at the .ab1 file?


Hi, guys, it seems i've found the culprit behind this, but i am not sure. when i got the new results of sequencing, i would give you feedbacks. thanks.

-doorsea-

doorsea on Sat Oct 29 05:27:39 2011 said:

allynspear on Thu Oct 27 14:00:33 2011 said:

I agree with phage434 that the sequence in its small format is difficult to read, but it looks okay. Is it just that the sequence is not what you expected? Just so that I am clear, did you send out the plasmids for sequencing this last time, or your PCR products?

phage434 on Thu Oct 27 13:03:19 2011 said:

The trace file is pretty difficult to read at the size you have uploaded, but from what I can see it looks as if you have good sequence. Are you certain there is a problem? What makes you think you have bad sequence? Can we look at the .ab1 file?
Hi, guys, it seems i've found the culprit behind this, but i am not sure. when i got the new results of sequencing, i would give you feedbacks. thanks.

I have got the right sequencing results. The culprit of failed sequencing before is the polymerase that was contaminated. After I change it with another one, the sequencing results are good. i was stuck in sequencing for almost 1 month. thanks for your patience

-doorsea-