I performed a PCR on some samples that looked good and only produced 1 band on a gel. After PCR purification with a Qiagen kit following normal procedures, I sent the samples out for sequencing. I get back my results and some feedback from the sequencing guy. He thinks there were some "PCR cleanup issues." The sequencing looks okay in some parts but has a lot of poor peaks that result in "n"s. Has anyone else run into this issue before?
As I side note: I have done PCR purification many times with this kit and have not run into this problem before.
Edit: Attached are some example ab1 file that were giving me trouble. One has nice peaks in a large amount but some mixed ones throughout.
Both files show signs of high background which is particularly poor in sample 1. The true signal strength is uniformly good ( above 1000 ) but in sample 1 the background signal gets stronger and is found over the entire sequence. The good signal strength and lack of dye blob suggests that the amount of sequence template,sequencing primer and purification of the sequencing reaction are good. It looks like the most likely reason for 2 sequences is the purification of pcr product from the primers has not worked well and some reverse primer has been left in the purified products. Check the age and quality of your clean up kit would be my first suggestion ( or try a different cleanup method). A useful check is also to look for any other possible binding site for your added primer within the template in case it is annealing in 2 places but since you have high background in both sequences and they are different templates then I think that this second option is unlikely,
You can get a result like this from old capillaries on the sequencer but not if it is well looked after so I also think that the cleanup procedure is the most likely source of the 2 sequences within 1 electropherogram
It happens sometimes. I would just do it again and send out. Do you use BigDye method? You have two PCR primers. You can try both direction (sequencing), either using the forward primer or the reverse primer for sequencing PCR. Sometimes, one primer will work better than the other one.
Selene Hess
can you attach one good sequence and one poor one please. There is often much to be learned from the basic data. Original .abi files please
Both files show signs of high background which is particularly poor in sample 1. The true signal strength is uniformly good ( above 1000 ) but in sample 1 the background signal gets stronger and is found over the entire sequence. The good signal strength and lack of dye blob suggests that the amount of sequence template,sequencing primer and purification of the sequencing reaction are good. It looks like the most likely reason for 2 sequences is the purification of pcr product from the primers has not worked well and some reverse primer has been left in the purified products. Check the age and quality of your clean up kit would be my first suggestion ( or try a different cleanup method). A useful check is also to look for any other possible binding site for your added primer within the template in case it is annealing in 2 places but since you have high background in both sequences and they are different templates then I think that this second option is unlikely,
You can get a result like this from old capillaries on the sequencer but not if it is well looked after so I also think that the cleanup procedure is the most likely source of the 2 sequences within 1 electropherogram
Result of Sanger Sequencing mostly depends on
1. Region amplified (some are easy and some difficult to amplify)
2. length of product (more length take more amount of PCR mix)
3. concentration of PCR product after purification (increase it)
Thank you Paul Rutland! I really appreciate your thorough explanation, especially since I like knowing what went wrong as opposed to just trying it again. I will try the things you have suggested!
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