PAN_DNASEQ

PAN: Protein and Nucleic Acid Facility

» DNA Sequencing
& Fragment Analysis

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Genomic Services

• Oligo Synthesis

• Gene Expression
& Bioanalyzer

• Real-Time PCR

• Pyrosequencing

• Next Gen Sequencing

• DNA Sequencing
& Fragment Analysis

Protein Services

• Protein Analytics

Located in rooms
B065 and B017

• SAMPLE SUBMISSION

FOR DNA SEQUENCING	FOR FRAGMENT ANALYSIS
Types of Services Sample / Primer Concentration DNA Sequencing Protocol Thermal Cycling Condition List of primers provided by PAN	Types of Services List of size standards provided by PAN

TYPES OF DNA SEQUENCING SERVICES (top)

Service	Description	Sample Submission Format
Full Service	DNA sequencing by PAN Reaction clean-up by PAN Sample run on instrument by PAN Submission deadline 12:00pm	*Click here for Template and primer concentration. Click here for a list of PAN provided primers.
Clean-Up & Run	DNA sequencing by customer Reaction clean-up by PAN Sample run on instrument by PAN Submission deadline 2:00pm	Click here for sequencing protocol and thermal cycling conditions.
Run Only	DNA sequencing by customer Reaction clean-up by customer Sample run on instrument by PAN Submission deadline 3:00pm

TEMPLATE and PRIMER CONCENTRATIONS (top)

Template	Size	Concentration	Volume (for 2 rxns)
PCR products	100bp-1000-bp	20ng/ul	10ul
	1000bp-2000bp	50ng/ul	10ul
	>2000bp	100ng/ul	10ul
Plasmids (dsDNA)	2kB-8kB	100ng/ul	10ul
Large Plasmids	8kB-20kB	200ng/ul	10ul
*BACs		500ng/ul	10ul
Custom Primer (for PCR and Plasmids)		10pmol/ul	2ul
*Custom Primer (for BACs)		100pmol/ul	2ul

Template should be provided as purified plasmid, BAC, or PCR product.
- We recommend using kits from Qiagen. .
Template and primer should be provided in water.
Primers should be 18-24 bases long, with Tm of 45-60°C
Premix template and custom primer prior to submission.
Please be aware the first ~20 bases of a sequence maybe unreadable.
All samples submitted to PAN are discarded after 2 weeks.

DNA SEQUENCING PROTOCOL (top)

Reagents	Big Dye (1/4rxn)	dGTP	BACs
Big Dye v3.1	2ul	2ul	6ul
Template: Plasmid (200-500ng) or PCR (10-100ng) or BAC (2-5ug)	5ul	4ul	8ul
Primer (10uM)	1ul	1ul	2ul
dGTP		1ul
DMSO (50%)		1ul
5X Buffer			4ul
Water	2ul
Total	10ul	10ul	20ul

THERMAL CYCLING CONDITIONS (top)

On ABI Thermocycler	Plasmid/PCR/dGTP		BAC
Initial Denaturing	96°C - 2min**		95°C - 5min
Denaturing Annealing Extension	96°C - 10sec 50°C - 5sec 60°C - 4min	} x25 cycles	95°C - 30sec 50-55°C - 15sec 60°C - 4min	} x100 cycles
HOLD	4°C - œ		4°C - œ

**For sample with possible secondary structure (before adding BigDye) denature sample at 96°C - 2min, chill immediately in ice bath. Once cooled add Bigdye and place back on thermal cycler for remaining cycling steps.
We recommend sephadex bead clean-up.

PAN PRIMER LIST (top)

Primer Name	Sequence
M13 Forward	5’- TGTAAAACGACGGCCAGT -3’
M13 Reverse	5’- CAGGAAACAGCTATGACC -3’
T7	5’- TAATACGACTCACTATAGGG -3’
Sp6	5’- ATTTAGGTGACACTATAG -3’
T3	5’- ATTAACCCTCACTAAAGGGA -3’

TYPES OF FRAGMENT ANALYSIS SERVICES (top)

Service	Description	Sample Submission Format
Clean-up & Run	PCR performed by customer Reaction cleaned-up by PAN Size standards added by PAN Sample run on instrument by PAN Additionl dilutions upon request	1ul-10ul of PCR product Record number on top of tube or plate
Run Only	PCR performed by customer Size standards added by PAN Sample run on instrument by PAN Additionl dilutions upon request	1ul-10ul of PCR product Record number on top of tube or plate

1ul of PCR product will be loaded with 0.25ul size standard and 10ul formamide, if additional dilutions are needed please let us know. RFU should be around ~1000-4000.
Clean-up of PCR reaction is not necessary, if requested sephadex G50 beads are used for clean-up.
All samples submitted to PAN are saved for 2 weeks.

PAN SIZE STANDARD LIST (top)

SIZE STANDARD DYE	FRAGMENT DETECTION DYES	FILTER SET	DYE SET
GeneScan 500-ROX	6-FAM, HEX, NED	D	DS-30
GeneScan 500-LIZ	6-FAM, VIC, NED, PET	G5	DS-33

GeneScan-500 is designed for sizing DNA fragments in the 35-500bp range and provides 16-single stranded labeled fragments of 35*, 50, 75, 100, 139, 150, 160, 200, 250, 250*, 300, 340*, 350, 400, 450, 490, and 500 bases.

(*) On capillary electrophorsis instruments, the 250bp and 340bp peaks are subject to temperature variations. Fragment analysis primer peaks can interfere with the detection of the 35bp peak. These peaks may need to omitted, optimize your analysis based on this information.