Dear Maria

as Guillermo told, with a single sequencing reaction can cover up to 500-1000 bp. Therefore to cover the entire plasmid you need to design many primers annealing every 500-700bp on the vector to be sure that the resulting sequences will overlap a bit and you will be able to re-built the entire sequence.

Of course to design all the primers in a single step you need to already know the theoretical sequence of your plasmid.

if is not the case you can use the sequence that you already obtain to design 2 new primers:

- a forward primer annealing close to the end ( eg at base 950 if your sequence covered 1000 bases) of the sequence

- a reverse primer Which annealing close to the beginning ( at base 50)

and send 2 new sequencing reactiib with this 2 primers to increase the sequence coverage and continue in this way with several step until you will cover the entire sequence.

good luck

Manuele

Your results are completely normal. You can't sequence more than ~1000 bp in a single Sanger reaction, even if it's really well optimized.

Normally, you only need to sequence your inserted region in the plasmid and not the entire construct. How big is your insert?

Dear Maria,

Once again as other said your results seem to be normal. In a single Sanger reaction the sequence lenght is abourt 1000 bp.

As Katie A Brunette asked you I will also ask which is the size of your ijnserted region?

Best of luck.

To sequence a 5Kb insert, you should expect to get about 5-6 sequence reads of about 800-1000bp overlapping at the ends. This is because Sanger sequencing can only read about 800-1000bp per reaction. You will need both a forward and reverse primer for each sequencing reaction. However, if you know of the sequence information for your plasmid ( containing the 5Kb insert which you wish to sequence), you can design a sequencing primer from it and get the first result ( about ( 800-1000bp), use the sequence information of the first result to design another sequencing primer which will allow you sequence the next overlapping section and continue to use the result of the second to design the sequencing primer for the next and so on! Once you are able to get the different sequence reads, you can then assemble using a software like Sequencher to get a consensus sequence which covers the whole range of your insert fragment (5Kb

I totally agree with Sarah Nanyiti, the reading limit of Sanger sequencing is about 850 to 1000 max ...its better to re design the primers F and R , within your sequence of interest and see how it goes .