PAN_DNASEQ

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Located in rooms
B065 and B017

• SUBMISSION REQUIREMENTS

The PAN Facility has three options of sample submissions for DNA sequencing.

Reactions NOT Completed – DNA sequencing is performed by PAN.
Reactions Clean-up – DNA sequencing performed by the client, sequencing clean-up performed by PAN
Reactions Completed – DNA sequencing and clean-up performed by the client.
*For DNA preparation, we recommend using purification kits from Qiagen.*

» Reaction Not Completed (top)

*PAN’s Recommended Concentrations

» Reactions Clean-up (top)

» Reactions Completed (top)

**PAN’s DNA Sequencing Protocol

Reagents	Big Dye (1/4rxn)	dGTP	BACs
Big Dye v3.1	2ul	2ul	6ul
Template: Plasmid (200-500ng) or PCR (10-100ng) or BAC (1.6ug)	5ul	4ul	8ul
Primer (10pmol)	1ul	1ul	2ul
DGTP		2ul
DMSO (50%)		1ul
Water	2ul
Total	10ul	11ul	16ul

**PAN’s Cycle Sequencing

On ABI Thermocycler	Plasmid/PCR/dGTP		BAC
Initial Denaturing	96° - 3min		96° - 5min
Denaturing Annealing Extension	96° - 10sec 50° - 5sec 60° - 4min	} 25 Cycles	96° - 30sec 50° - 15sec 60° - 4min	} 100 Cycles
Hold	4° - œ		4° - œ