sequencing (sanger method)

[limah]

hi,
upto how many base pairs can the sanger or chain termination method sequence? i thought it was upto about 2kb but recently i tried to sequence a 1.4 kb pair fragment, but i didn't obtain the full length sequence i only obtained about 1kb of the sequence ??????

[olgagog]

Hi.
Sequencing with just one primer, it is very difficult to get to sequece more than 700pb. It also deppends on the quality of the DNA you are using. And it also deppends on the concentration of the terminators, and the length of the run. How many primers are you using for sequencing?

[lalarka]

hi,
upto how many base pairs can the sanger or chain termination method sequence? i thought it was upto about 2kb but recently i tried to sequence a 1.4 kb pair fragment, but i didn't obtain the full length sequence i only obtained about 1kb of the sequence ??????

It is quite difficult to get quality reads in excess of 1KB using current standard procedures. The primary driver for good sequencing is cleanliness of template and quality of primer.
My company routinely performs 10,000 reactions a day on difficult template clones and we see ~800 cLOR (clean Length of Read). You should also figure that the first 20-30 bases will be compromised and that anything toward the end should not be trusted without manually verifying the base calls.

If you want publication quality reads, plan on doing a primer walk every 500 bases and then assemble the contigs.

Here's an example of one of my reads. You can see how the initial 20 bases are poor, then the poly G 10 stretch, followed by about 600 visible bases. The read continues with another 200 Q20+ scored reads. They're just on the next page.


Good luck,
Lance Larka
Oblique Bio, Inc.
http://www.obliquebio.com

[relaxin]

You can synthesize primer based on the sequence you obtained and sequence further downstream. Also you can sequence the other strand. Reliable sequence data need sequencing both strands.