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Plasmid sequencing failed why?


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10 replies to this topic

#1 Sagaguchi

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Posted 28 June 2011 - 06:55 AM

Hi everyone,
I did cloning of about 500bp insert into pESC-leu plasmid. I get correct band size into DNA gel after diagnostic digest with NotI and SalI. but unfortunately, when I send my plasmid (Qiagen purified)for sequencing it failed. I used reverse Primer from vector a liitle below the insert site. Concentration of DNA is about 151 ng/ul. and 260/280 ratio is about 1.9 so DNA is quite pure. I am wondering what could be possible reason for sequencing failed?
Any suggestion will be appreciated.

Thanks

#2 andrea_UNSAM

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Posted 28 June 2011 - 12:18 PM

hello, it only realises it to the quantification by nanodrop? if he is thus, first you should ran a gel and to see that really your DNA is there. Of another form, if you quantify without seeing, for sure you measuring DNA genomic, or even fragments of your plasmid, that as it is DNA, is going away to quantify. Test to ran a gel if you have not done it, and soon quantifies by nanodrop to confirm that what you measure is your plasmid. That happend to me before...:(

#3 Sagaguchi

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Posted 28 June 2011 - 01:36 PM

Hi andrea,
I have run the gel already. and did the diagnostic digestion with restriction enzyme and got right size band which means that I have my plasmid in there. And quantification was done by sequencer. I never done nanodrop. can you explain a little bit more about it?
Many thanks for reply

#4 phage434

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Posted 29 June 2011 - 04:49 AM

You haven't told us what happened to your sequencing reaction. It's usually a simple setup. The amount of DNA matters (not too much, and also not too little). You need a single primer (not two!) at the recommended concentration. Other than that, the reaction almost always works. Did you get bad data, no data, no priming?

#5 Sagaguchi

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Posted 29 June 2011 - 05:58 AM

You haven't told us what happened to your sequencing reaction. It's usually a simple setup. The amount of DNA matters (not too much, and also not too little). You need a single primer (not two!) at the recommended concentration. Other than that, the reaction almost always works. Did you get bad data, no data, no priming?

Actually, I send my Plasmid DNA for sequencing to other department. They measure the nanodrops and everything. I used only one primer i.e Reverse one. I got no data and they write in report that its because of no or very low HQBs (High Quality Bases).
Thanks for response

#6 phage434

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Posted 29 June 2011 - 05:21 PM

I'd say it's likely that your primer is not binding to your plasmid. You could test this by doing a PCR reaction with the sequencing primer and any other primer that will yield a relatively small DNA fragment. If this works, then you should try sequencing using the other primer, and work from there.

#7 Sagaguchi

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Posted 29 June 2011 - 08:34 PM

I think to do PCR with this primer is a good option. Thanks a lot.

#8 greenhorn

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Posted 03 September 2011 - 06:31 PM

Hello everyone,

I am having a similar problem with my sequencing results from our sequencing unit.
I screened my colonies by PCR before selecting them for doing my minipreps, which I then sent in for sequencing. I have been using exactly the same primers for the PCR screen (two in the insert overlapping strategies spanning each the corresponding restriction site into the backbone) as for the sequencing. In the PCR I had perfect bands of the correct size.
In the sequencing in contrast, I suddenly don't get any signal with the primers in the insert. The primers from the pLKO backbone give me a signal of the beginning of the insert from each restriction site, but the rest of the insert is completely missing (as if I had cloned primer dimers or so).

Does anyone have an idea what could be a reason for these strange results ?

I also did some other cloning of a CMV promoter into another backbone. Here, I have strange sequencing data as well. In every construct, a similar but mostly a little different part of the insert (somewhere in the middle) seems to be missing. But in one of the constructs, when sequenced with another primer from the other side (which did not work in most of the constructs in spite of the fact that the sequence was there with another primer...), most of one of the missing parts is suddenly present.

Is there any sequencing problem, which could explain those difficulties all together?

Thank you very much!!!!

#9 phage434

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Posted 03 September 2011 - 07:00 PM

It's very hard to know without more detail. How are you examining the sequence data? Do you look at the trace file, or at the bases? How are you comparing the sequencing results with the expected construct?

#10 greenhorn

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Posted 03 September 2011 - 09:47 PM

I am using the Staden Package (gap4) to look at the trace - but the alignment didn't really work, so I additionally copied the sequence bases into a word file where I looked for the sequence parts separately. I compare with a file where I assemble everything as it should be according to the cloning strategy.
The trace files in the pLKO constructs have a good signal - without the good, long PCR bands I'd say I cloned primer dimers...(found out they have 3 overlapping bp, but I used the same strategy for another backbone and it worked well).

In the CMV promoter sequencing the signal is very poor in general...

I hope this helps! Thank you very much in advance!!

#11 phage434

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Posted 04 September 2011 - 07:12 AM

It's very hard to know what is happening without a lot more detail and some experimentation. The sequencing primer site could be missing from your "correct" clone which shows the RE digest pattern you expect. The sequencer could then be sequencing some other plasmid contaminant present in your sample (or carryover from the ligation). I'd re-transform your DNA, streak for single colonies, miniprep and try again. You could also try some additional primers sequencing from your insert into the plasmid.

You might be better off making the construct again and throwing these away.




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