Hello everyone,
I've started doing molecular biology, rather recently. So, I've spent some time to obtain my first sequences at ABI platform and all of it were perfectly readable wheteher seq. analysis soft or bioedit. Meanwhile, the last sequences I had consist of unreadable chromatograms as well as lack of letters, although chromatograms were visible but not from the start (initial) position (attached picture). As far as I guess, it should be possible to move initial reading site of chromatogram somehow but I could not do it by my own.
I would really appreciate any suggestions solutions etc. Otherwise, if I have only two options either put letters by myself or perform sequencing again?
P.S. can anybody give some assumptions what is it related to and how to avoid it at further?
Warm thanks
By the way, I've tried to read it by chromas but nothing happened, as well. Attached is chromas file.
Hi,
please look over here:
http://chemistry.umeche.maine.edu/CHY431/BioEdit.html
http://www.sfu.ca/~donyang/arch335/Protocols%20for%20BioEdit.pdf
How long is the sequence that you expect? I see the beginning is quite noisy and maybe at the end the peaks are clearer, nevertheless i find it weird that the software is not showing you the letters in the peaks that seem with better quality and signal. I guess it is not because there is an option to move the reading window of the chromatogram but because the sequence quality, this is why i am asking you how long is the sequence that you expect? if then at the end you see better quality? how are you cleaning your pcr product?what is the concentration of the purified product?
the average lenght of obtained sequences about 600 nt, the technique to obtain this picture included following steps:
1. rt-nested-pcr to obtain long product which is about 1000 nt
2. then performing PAGE with 10 mkl of product with subsequent cutting from gel under UV light
3. Gel was placed to tubes and covered by ddwater (over 20 mkl) then left on 12 hrs at +4 C
4. 2 mkl of water was taken and placed to 2 mkl big dye terminator mix, plus 2 mkl of each sequencing primer into different tubes. So, final mixes consist of 6 mkl (2 product, 2 BDT, 2primers). Then seq. reaction was performed.
5. final reaction product was fully (6 mkl) placed into wells and cleaned by XT terminator (10 mkl) and SAM solution mix (45 mkl) by orbital shaker and then it was placed to ABI. At least final step dilution was 1/10.
Do you think the pcr product is to be diluted? So, does this amend need to be performed either after pcr step, elution at ddwater or after sequencing reaction?
The obtained chromatograms are quite quality at whole sequence because I compared it with previous which were auto analyzed by bioedit and I have't found any differences in peaks and noise, except empty start region.
Thank you
so, if i am understanding correctly, these lasts sequences show the correct length (600 nt) but not the letters even when the sequence is less noisy? Well, maybe you can repeat the PCR and the purification to see if still you produce the same kind of files without letters... maybe the issue is more technical with the sequencing machine?
I have never cut PAGE gels, if i have many bands i cut a normal agarose gel and purify or clean the product after the PCR... I do think is a good idea to check the concentration of the purified product to see if you have enough. If it is too concentrated or too diluted the reaction fails. We usually send to a company and they required a minimun concentration so that sequencing reaction is efficient.
You can try to check Your sequences in MEGA software. MEGA is provided free for use in research and education. If the results are same, You have to perform sequencing again.
The main reason of unreadable chromatograms can be that DNA template is of poor quality or concentration is too low/high. As Diana wrote, a good idea is to check the concentration of purified product before sequencing.
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