Using an ABI 3130xl:
Let's say you want to sequence the CO1 region for the same individual.
For the amplification: you design two CO1 regions: One region is ~550bp longer than the other but they both use the SAME forward primer.
You use the SAME recipe for cycle sequencing and the same run parameters to sequence all primers (you want to be able to make a contig for both fragments, so in total, you want 4 sequences, 2 for each fragment--even if the same forward primer is used for both amplifications).
Your sequences yield good results BUT: the shorter fragment gets a better read than the longer fragment. The sequences from the longer fragment lose resolution sooner than the sequences from the shorter fragment. Hence, you can't get a contig from the longer fragment sequences.
Could this be due to the length of the fragments and why would the same primer lose resolution sooner?!
Would you try different parameters (in recipe or run parameters) to sequence the longer fragment?
Thanks again!!
D.
Let's say you want to sequence the CO1 region for the same individual.
For the amplification: you design two CO1 regions: One region is ~550bp longer than the other but they both use the SAME forward primer.
You use the SAME recipe for cycle sequencing and the same run parameters to sequence all primers (you want to be able to make a contig for both fragments, so in total, you want 4 sequences, 2 for each fragment--even if the same forward primer is used for both amplifications).
Your sequences yield good results BUT: the shorter fragment gets a better read than the longer fragment. The sequences from the longer fragment lose resolution sooner than the sequences from the shorter fragment. Hence, you can't get a contig from the longer fragment sequences.
Could this be due to the length of the fragments and why would the same primer lose resolution sooner?!
Would you try different parameters (in recipe or run parameters) to sequence the longer fragment?
Thanks again!!
D.
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