General Description of Instruments Automated DNA sequencing is perfomed with both gel slab (LICOR) and capillary electrophoresis (Beckman) machines. Both utilize Sanger dideoxy cycle sequencing protocols using single and double stranded templates. Up to 16 slab gel samples can be run at a time. Direct sequencing from purified PCR product can be perfomed on the CEQ 8000. Sequencing runs can be run in combination with genotyping samples in a 96 well format.

   

LI-COR 4000L and 4200  IR2:  The 4200 IR2 is equipped with a fluorescence microscope and solid-state laser diode which can detect infrared dye-labeled primers at 700 and 800 nm.  The 4000L is a single laser machine which detects at 800 nm.  It is possible to run bidirectional reactions in one set of tubes to sequence both strands of the target DNA at the same time. Typical runs during the week are on 41 cm long (0.25 mm thick), 6% Long Ranger polyacrylamide gels, which run at 50 C and 35.0 mA overnight for approximately 12 hours. These gels typically give reliable read lengths between 600-800 bases. Longer reads are possible with 66 cm long, 4% gels, which run at 45 C and 25.0 mA for a minimum of 24 hours. Depending on template length and quality, between 900-1,200 bases are possible.

     

Beckman CEQ 8000: This is a versitile moderate through-put machine capable of processing 8 sequencing samples in just under 2 hours.  Read lengths beyond 700 bases are possible. pUC18 control templates routinely analyze to 720 bases with over 98% accuracy under default running conditions. Removal of unincorporated dye terminators, primers, and salts using magnetic beads (CleanSeq, from Agencourt), is fast and very reliable. Turnaround time is generally 24 hours.  Both sequencing and fragment samples can be run on the same 96 well plate.  Contact the lab for further details.

Please send questions and comments about this site to the site administrator