I have had problems sending some samples to sequence the COI and 18S gene. The results are not good. Seeking to obtain sequences: COI =700 bp approx and 18s = 1069 bp approx.
Although my gels exhibit relatively good bands, the sequences obtained have a low quality (very poor quality). In the attached document I describe in more detail the processes by which I got these bands.
Might it be the low quality of DNA?
A bad PCR or electrophoresis protocol?
Improper handling of the samples at the time of delivery?
Any advice?
(If you require more detailed information, please request it and I will gladly facilitate this)
Usually if you get a bad sequencing can you repeat the extraction again... because the quality of DNA affected on the quality of sequencing.
when you purify the PCR product from gel for DNA sequencing you have to be careful. You should not try to take as much as you can because it may add impurities of phenol or other reagent. if your gene is in a vector and you are sending sample after plasmid miniprep then this chances of impurity are very less. But in both the cases after you first check your DNA sample in Agarose gel to check if band is clear or not.
Thank you very much to all.
Artur Burzynski : Thank you very much for the suggestions, I hope to answer your questions properly.
* The PCR is purified by the company that sent sequenced (Macrogen), I do not know the technique used.
* The initial concentration is measured after extraction using a Nanodrop, giving values between 1000un/ul 300ng/ul and then made dilutions (50ng/ul), finally using 2ul of DNA sample by PCR in a total volume of 25ul by reaction.
* We have previously used the services of Macrogen and had not arisen problem.
Laura Collopy: I hope that if
Note: I add some files from the sequencing. The LCO appointed correspond to COI and others are 18s.
Thank you very much
*I'll check the concentration at which sending my samples.
*I have reviewed the sequences or fragments of these but have no meaning when compared to previous sequences had good results.
*Resequencing of my samples was performed but no improvements.
I have checked the primer sequence for COI. I think there is problem in primer design. You may follow the general principles of primer design. The sequence should include 20 bp (17-23bp), with Tm value 62-65C (>55C) and GC value should be 50% (40-60%). Hope, you will get good results.......
Hi Rodolfo,
I use to send my samples to Macrogen for Sanger Sequencing. Sometimes sequencing fails for no apparent reason. I mean DNA amplicons that have been successfully sequenced in the past fail for some concrete DNA samples (that show nice amplification bands in the agarose gel). In my opinion that excludes the possibility of wrong primer design, sample purity or concentration. But when sending a new aliquote of the SAME previous PCR reaction sequences are perfect. Sincerely I think that sometimes there's something wrong with Macrogen.
Macrogen offers this special service called "Standard Sequencing" where the client sends the primer and the sample in separate tubes. Then they try different sample and primer concentrations in order to find the right ones. The theory is fantastic but when analyzing the same sample using both basic and standard sequencing protocols the results are deceiving. Sometimes the basic one works better (!)
Does anybody had any experience of this kind?
Hi Celia Gracia,
I sent samples (PCR product) to Macrogen for COX2 (FM66 and FM58 primers) and ITS (ITS4 and ITS6 primers).
Product size: COX2 (~600pb) and ITS (~850pb)
fungi DNA was extracted with column.
PCR product were good. band single.
But, the chromatograms were bad.
My second sent, the primer FM66 was good but primer FM58 was bad.
FM66, only 300pb good quality.
ITS 4 was bad and ITS6 was so so (184pb - good quality)
My third sent, the primer FM66 was bad and primer FM58 was good.
ITS4 was bad and ITS6 was so so.
I think that Macrogen's technician had problems with PCR product purification.
I don't know, why it can happen.
I previously sent samples for the same primers and the results were good.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA