DNA Requirements for Sanger Sequencing

Mix your template (5 μl) and primer (1 μl of 3.2 μM) according to the specifications below.

DNA Type	Template Length	Template Amount	Primer Amount	Premixed Volume
Plasmid (vector + insert)	1 kb – 10 kb	200 ng	3.2 pmol	≥ 6 μl/reaction
	10 kb – 20 kb	400 ng
PCR products & DNA fragments	100 – 500 bp	5 – 20 ng
	500 – 1000 bp	20 – 50 ng

* For large DNA such as plasmids over 20 kb, Lambda, Cosmids, and BACs, please contact us for further details before submitting for sequencing.

 

 Note:

If you want to submit your template and primer in separate tubes, provide 10 μl of template and 10 μl of primer (diluted to 3.2 μM). This is double the amount of template and primer volume that we actually need so that we can rerun samples if there are any problems. Your templates and primers will be kept for one week in case you need them returned back to you.

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 Supplied Primers

ASGPB provides the primers listed below at no additional cost:

Primer	Abbreviation	Sequence (5′ to 3′)
M13 Forward	M13F(-20)	GTA AAA CGA CGG CCA GT
M13 Reverse	M13R(-27)	CAG GAA ACA GCT ATG AC
T7 Promoter	T7	TAA TAC GAC TCA CTA TAG GG
T7 Terminator	T7 ter	GCT AGT TAT TGC TCA GCG G
T3	T3	AAT TAA CCC TCA CTA AAG GG
SP6	Sp6	ATT TAG GTG ACA CTA TAG

The following are some tips to help design your custom primers:

The primer should be at least 15 to 20 bases from the insert

The primer should be between 18 to 25 bases long

Aim for ~50% GC content, with the Gs & Cs evenly distributed throughout the primer

Avoid runs of identical nucleotides

The Tm should be 55°C to 75°C

Use software to check your primer for any dimers or hair-pin loops that may be formed

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 Sample Submission

Fill out the Sequencing Order form and submit with your samples to:

ASGPB

University of Hawai‘i at Mānoa

1800 East-West Road, Life Sciences Building 320

Honolulu, HI 96822

Drop off samples Monday through Friday between 9:30 a.m. to 5:30 p.m.

         *If you are shipping samples to us, use the following mailing address:

         ASGPB

         University of Hawai‘i at Mānoa

         3190 Maile Way, St. John 101

         Honolulu, HI 96822

 

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Data Delivery

You will be notified by e-mail with your finished sequences.

You should receive the results in one or two business days unless there are unforeseen delays. In that event, you will be notified as soon as possible after the samples arrive at the facility.

The results for each sequence will be provided in abi format files. The ABI file (.abi) contains the nucleotide sequence, electropherogram, and other information about the run conditions. This file requires software such as Chromas, 4Peaks, Geneious, etc to view it.

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Contact

If you have any questions or would like more information about our other services, please contact Shaobin Hou or Jennifer Saito.

Email: asgpb07@gmail.com or asgpb@hawaii.edu

Phone: (808) 956-8039