Sanger DNA sequencing

DNA Sequencing

• The automatic DNA sequencing developed by Applied Biosystems uses the methodology of Sanger, which is based on the use of a thermostable polymerase that allows a cyclic sequencing reaction.
• The product of the reaction is separated using a capillary electrophoresis. Fragments are labelled using 4 fluorochromes (one per nucleotide). These fluorochromes can be incorporated either into the primer, “dye primer kit”, o into the ddNTPs o terminators, "dye terminator kit".
• The DNA to be sequenced must have the highest quality, and it could have been obtained from: PCR products, plasmids or phages. The primers for the cyclic sequencing can be either those specific primers for each PCR product or the universal primers of plasmids or phages.
  
  

DEVICE

Sequencer ABI 3130XL

KIT

BigDye 1.1 ,  BigDye 3.1

SAMPLES

192 samples/day (2 plates)

M13univ: 5'-GTA AAA CGA CGG CCA GT-3'

M13rev: 5'-CAG GAA ACA GCT ATG AC-3'

SP6: 5'-GAT TTA GGT GAC ACT ATA G-3'

T7: 5'-TAA TAC GAC TCA CTA TAG GG-3'

T3: 5'-AAT TAA CCC TCA CTA AAG GG-3'

T7term: 5'-GCT AGT TAT TGC TCA GCG G-3'

CAPACITY

16 simultaneous readings up to 800-900 pb

RESULTS

Results are sent by electronic mail

ACTIVITY

CONDITIONS

Electrophoresis of samples previously sequenced by users.

The dry pellet of DNA must be brought within tubes of 1,5 ml or 0,5 ml, or resuspended within 96-plate.

Sequencing reaction and electrophoresis.

DNA must be diluted in water at a rough concentration of: 20 ng/µl for PCR and 100 ng/µl for plasmids.

Also, the primer must be diluted in water at  a concentration of 3,2-5 pmol/µl.

Preparation and/or purification from a culture plate (plasmids) or from PCR reactions

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Other services trough a previously established collaboration or agreement

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