PAN_DNASEQ

PAN: Protein and Nucleic Acid Facility

» DNA Sequencing
& Fragment Analysis

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Genomic Services

• Next Gen Sequencing

• DNA Sequencing
& Fragment Analysis

• Single Cell Genomics

Protein Services

Located in rooms
B065 and B017

• SAMPLE SUBMISSION

DNA SEQUENCING	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	Purified DNA template sample submitted by customer Custom primers should be premixed in tube BigDye DNA sequencing reaction performed by PAN Requested PAN primers will be added DNA sequencing reaction clean-up performed by PAN Sample run on ABI 3130xl by PAN Submission deadline 12:00pm Labeling - please write Record Number(s) on tube(s).	*Click here for Template and primer concentration. Click here for a list of PAN provided primers.
Clean-Up & Run	BigDye DNA sequencing reaction submitted by customer DNA sequencing reaction clean-up performed by PAN Sample run on ABI 3130xl by PAN Submission deadline 12:00pm Labeling - please write Record Number(s) on tube(s).	Click here for sequencing protocol and thermal cycling conditions.
Run Only	BigDye DNA sequencing & clean-up performed by customer Sample run on ABI 3130xl by PAN Submission deadline 12:00pm Labeling - please write Record Number(s) on tube(s).

TEMPLATE and PRIMER CONCENTRATIONS (top)

Template	Size	Concentration	Volume
PCR products	100bp-2000-bp	1-10ng/ul	10ul
	>2000bp	10-20ng/ul	10ul
Plasmids (dsDNA)	2kb-8kb	50-100ng/ul	10ul
Large Plasmids	>10kb	100-200ng/ul	10ul
Custom Primer		5uM (5pmol/ul)	2ul

Template should be provided as purified plasmid or PCR product.
- We recommend using kits from Qiagen.
Premix template and custom primer prior to submission.
- One template + one primer sample tube.

Template and primer should be provided in water.

Primer should be 18-24 bases long, with Tm of 45-60°C
Please be aware the first ~25 bases of a sequence may be unreadable.
10ul-12ul total volume is requested to allow for resequencing if necessary
All samples submitted to PAN are discarded after 2 weeks.

Sorry, we are no longer accepting BAC, cosmid, or bacterial genomic DNA samples.

DNA SEQUENCING PROTOCOL (top)

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

THERMAL CYCLING CONDITIONS (top)

On ABI Thermocycler	Plasmid/PCR/dGTP
Initial Denaturing	96°C - 2min**
Denaturing Annealing Extension	96°C - 10sec 50°C - 5sec 60°C - 4min	} x25 cycles
HOLD	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 Additional 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 Additional 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 between 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 discarded after two 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
CUSTOM DYES or KITS	PLEASE CONTACT PAN	INQUIRE	INQUIRE

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 be omitted. Optimize your analysis based on this information.