Amount, concentration and volume of PCR fragments for sequencing

Conditions required for sequencing of PCR products:

 size of fragment
(bp)		  DNA amount
required per reaction
(ng)		 required
DNA concentration
in sample (ng/µl)		  required
sample volume
(µl)
100 10 10 5
200 20 10 5
300 30 10 5
400 40 10 10
500 50 50 5
600 60 50 5
700 70 50 5
800 80 50 5
900 90 50 10
1000 100 50 10
1200 120 100 5
1500 150 100 5
2000 200 100 5
2500 250 100 10
3000 300 100 10
5000 500 100 10

Please note that there are a number of important factors for successful sequencing of CLEAN and primer-free PCR fragments:

  1. ensure that there is a single band (or, gel-prurify a single fragment to remove other amplification products),
  2. do some basic checks to verify that the proper product was amplified (prove BOTH primers were essential, restrict the fragment at a diagnostic site if possible),
  3. make sure that the primers are completely removed from the PCR fragment (we recommend EXO-SapIT from A)
  4. ensure that you provide a sufficient amount of PCR fragment (template) for the sequencing reaction see table above - quantification of the DNA via a gel is recommended),
  5. ensure an appropriate concentration of the template DNA which fits the needs of the sequencing reaction setup (see table above),
  6. provide volumes which allow exact pipetting.