Help: ABI 3130xl and long fragment sequences.

[dmrodz]

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.

[dmrodz]

For sequencing, you use the same primers you used for PCR.

[SeqAA]

It might be the longer fragment is not denaturing completely before the cycle sequencing reaction. Try adding a 5-10min denature before you start your cycle conditions. This kinda helps for everything, and is a good place to start.

Some people denature first, then add their master mix to the sample. I was always too lazy for this, and it seemed to work fine.

[pmiguel]

Hi dmrodz,
Can you give a little more information? What read lengths are you getting in each case? What signal strength do you see? (S/N% for the A,C,G,T) How much big dye and how many cycles are you using? How much template are you adding to the reaction? How do you quantitate the template?

--
Phillip

[barrmur]

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.

An old post I know but your problem is likely due to the concentration of either your template or primers. If the template concentration is to high the primers are used up during cycle sequencing very quickly and as a result longer dye terminated products are not as plentiful for electrophoresis. The same problem exists if the primer concentration is to low. For template try 1ng/ul/100bp of product and make sure primers are in excess.

[Atypus]

3130xl DS-33 standards

Hello. I would like to ask you why or on what purposes DS-33 GeneScan Installation Standards are suitable? Are these standards required for direct sequencing or only GeneScan 500 Liz Size Standard is required? Thanks