Bad sequences - (Nov/23/2006 )
Got some sequences yesterday that is about 700 bp long, but the first 400bp is very bad and you cant read it. the last 300 is really good. Do someone know why the fist part of the sequence is like this.
I use the following conditions for the 10ul sequencing reaction
Big dye 1ul
5*buffer 2
primer(3,5uM) 1ul
template 40 ng
water
Here is one of the sequences.
NNNNNNNNNNNNNNNNNNGANNNNNCGNNNNNNNCCCACAAC
AANNNNNGNGNNNGNNNNGNNNCNNAANNNNNNNNNNNNNNN
NNNGNANGCNNNNNNNNNNGAAGTACTCCAAGGGNNANANCTG
ANCNCNNGGNTCNTCCTCTCANNNNNNNNNNNNNNNGNNNNNN
NNNNNNNNNTACNNNNNANNCAGANNNNNAACCCTNCNNNNNN
NANNNNNATTGGCTTACTATCNANNNNNNNTGGTGGCTGANGGG
GTCNNNACCANNNNNNNNTNNNNAANGNNNNCNCCNNNNCNTC
NNNNGNCCACNNNNNNTGANNNNNGCTGATCCTNNNNNNTNNN
NNCCNNNNNGNCCNNCNCNCCCTNNNNNNNGACTTTANNNNNA
NNNNCTCANCATTCCNAATTTTNAAAGNGNANAAANNNACNNCGN
NCAATGCCCTCGNNNNNNNNNGNANAANAACTCCTGNCNNNNGA
NNCCTAGCACCCCTGGTTTTACTTTCTCTCGGCGGCCTTCCTCCC
CTTACCGGCTTCATGCCCAAGTGATTCATTCTTCAGGAACTGACTA
AACAAGACCTTCCGGCACTTGCCACCCTCGCCGCTTTAACTGCCC
TCTTGAGTCTCTACTTCTACTTGCGCCTCTCGTATGCAATAACCCT
CACAATATTCCCCAACAACCTAATTGGTGTCACTCCCTGACGATTC
TACTCCCCCCAATTTACACTTCCCCTCGCCGTCTCCACTGCAGCA
ACCACCCTTNNNNNCAN
Did you clean up your template prior sequencing?
How about your sequencing result of the reverse strand?
The pcr-product were run on a agarose gel, and i was using a gel extraction kit to isolate the DNA.
sounds like your primer is binding in two places of your template. You could try increasing the annealing temperature of your sequence reaction which may prevent binding to the second site and thus improving your sequence. Or perhaps designing a new primer with the 300 bp sequence you have and sequencing outward.
It's essentially impossible to tell without looking at the electropherogram. I doubt it is primer binding in multiple locations, as that would produce peak doubling throughout the sequence. My guess, with very little to go on, inaccurate location of the run start by the base calling program. Poor cleanup of the sequencing reaction might also do this.
Its true there isn't enough information, but if the template is a PCR fragment then binding of the primer in two positions would give the result that is being stated here. For example, the primer binds at the front of the PCR product and then 400 bp from the end of the PCR product. The first priming site would give polymerised products up to 700 bp or more, the second priming site would give a maximum 400 bp of polymerised products. When run on a polyacrylamide sequencing gel, only the first 400 bp would give double peaking..not the remaining 300.
Good point. I hadn't thought of that.