I'm working on Sanger sequencing using Bigdye V.3.1 and purify the sequenced product by DyeEx 2.0 kit. The chromatogram was fine and readable, however, it always contains some noise background and I need to minimize it. Is the ethanol precipitation by 125 mM EDTA and resuspend in Hidi is a better solution for sequenced product purification? since I have found some storage solution from the DyeEx column eluted with the purified-sequenced product.
Thank you.
I personally had the same problem.I did 2 things:
1. I stopped purifying the product in that way but instead I dilute the samples using nuclease free water according to the thickness of the band when run on the Gel.
2. Another issue I had was also with the annealing temperature, it was too low and so was binding in non-specific areas and I was getting more than one product, so try increasing your annealing temperature.
Goodluck
After PCR I use gel extraction for purification, therefore, there is no carryover primer after this step.
can we see the .abi sequencing file for one of your sequences please? it may help identify the problem
Dear Natthaporn
Thank you for the .abi file. This is good even spaced sequencing with well cleaned dye blobs run with in date gel matrix
so where is the background coming from?
Some possibilities of high background are dirty pcr amplification so 2 sequences are present. For reasons later I do not
think this is the case.
Incomplete removal of on primer so there are traces of 2 primers present in the sequencing reaction. If the product is
gel purified this is not the case.
Sequencing with a primer with n-1 contamination gives this effect but is unlikely as there are no completely clean peaks
where 2 or 3 peaks of the same base run together there should be no background
Old or dirty capillaries on the sequencer..check other sequences from column number 2 to see if others have high background
but low probability.
Looking at the data the raw signal intensity is mainly less than 150 ( 88-151) for the bases, It is also clear that the
sample loads slowly and the capillary current is unstable for about the first quarter of the run. This coincides with the
first 100-110 bases of the sequences being lower intensity than the later bases.
Mainly for this reason I think that you have too much salt in your sequenced sample. In this situation when you turn on
the loading voltage the salt loads in preference to the dna which is a larger molecule so you are not loading enough sequencing product.
The signal strength is low and the software pulls the sequence and also the background up making the electropherogram look dirty.
You may need to check your elution buffer/water used to get your purified sequenced product.
If you still have your sequenced samples run the plate again under the same conditions because the first run loading will
have removed much of the salt present and more dna will load second time round for a stronger signal.
If you cannot do this then try increasing your loading time from 13 secs to 20 secs unless you can see where to get rid
of a bit more salt from your sequenced sample ( possibly a 70% ethanol wash or increasing the volume of the ethanol wash)
Hope there is something here that helps
Paul
Dr Takpho, I agree with Paul. Additionally, I suggest you to check the calibration of the instrument. It looks to me that the detector is not able to correctly define the signals due to low base call. You might have to modulate the base calling paramaters. Apart from the initial and final Ns, rest of the reads appear good and also show very good match in BLAST search. Just get the instrument settings and calibration re-checked by the ABI experts. I think this will help you.
Best wishes,
SD
Thank you very much. I’m also thinking about this issue since the machine is quite old.
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