Sanger Sequencing

Sanger sequencing services are provided using the Applied Biosystems 3730xl DNA Analyzer. These instruments utilize a one-capillary cycle sequencing process with refined, four-dye fluorescent labeling methods and a real-time scanning detector. The biochemistry of the one-lane sequencing is similar to Sanger-based methods.

Samples should be provided pre-mixed with primer in 1.5ml tubes (please do not send PCR tubes). In each tube, mix 6 ul of primer and 6 ul of template. Use only one primer per tube. To achieve the best sequencing results, start with the following concentrations prior to mixing:

Primer: 1 uM for plasmids and PCR products, 4 uM for BAC/PAC/Cosmid

Purified PCR Products: 3.0ng/ul/100bp

Plasmid DNA: 0.2 ug/ul, with a 260/280 ratio between 1.75 and 1.85. 260/280 ratios above 1.9 or below 1.7 will not work.

BAC/PAC/Cosmid DNA: 0.5 ug/ul

If using the primers available at DAF, please bring 6 ul of template in a tube and indicate which primer should be added. Please clearly indicate which primers need to be added to the tubes. There is no charge for using our primers.

Tubes should be labeled with the order tracking number and the number indicating the sample’s position in the order, i.e. if it is the first sample in the list, place the tracking id and the number 1 on the lid of the tube. If submitting 24 or more samples, you may put them into a 96 well plate PCR plate (in column order) and label the plate with the tracking ID.

The customer is responsible for quantitating DNA and mixing with primer. Custom primers should be premixed with the sample prior to being dropped off. If you are using a standard sequencing primer, please indicate the appropriate primer on the order form and we will add the primer when we receive your samples.

The service provides standard sequencing primers for several vectors. Custom primers should be provided by the user and premixed with the template. Primers can be ordered through our facility using  one of our portals as well.

PCR Support is available for customers who don’t have the resources to amplify samples in their own lab.  Pricing is dependent on sample number and project complexity.   Please inquire.

Insert walking and other types of specialized sequencing support are also available.   Please inquire.

BGH Reverse	TAG AAG GCA CAG TCG AGG
CMV-Forward	CGC AAA TGG GCG GTA GGC GTG
EBV-RP	GTG GTT TGT CCA AAC TCA TC
KS	CGA GGT CGA CGG TAT CG
M13 Forward	TGT AAA ACG ACG GCC AGT
M13 Reverse	CAG GAA ACA GCT ATG ACC
Myc	GCA TCA ATG CAG AAG CTG ATC TCA
Oligo dT-A	TTT TTT TTT TTT TTT TTT TTT TTT TA
Oligo dT-G	TTT TTT TTT TTT TTT TTT TTT TTT TG
Oligo dT-C	TTT TTT TTT TTT TTT TTT TTT TTT TC
Oligo dT-N	TTT TTT TTT TTT TTT TTT TTT TTT TN
pDONOR-FP	TAA CGC TAG CAT GGA TCT C
pGEX3	GGA GCT GCA TGT GTC AGA GG
pGEX5	GGC AAG CCA CGT TTG GTG
SP6	GAT TTA GGT GAC ACT ATA G
SK	TCT AGA ACT AGT GGA TC
SV40pA-R	GAA ATT TGT GAT GCT ATT GC
SV40-promotor	TAT TTA TGC AGA GGC CGA GG
T3	ATT AAC CCT CAC TAA AGG GA
T7	TAA TAC GAC TCA CTA TAG G
T7 Terminator	GCT AGT TAT TGC TCA GCG G
U6 (LKO.1)	GAC TAT CAT ATG CTT ACC GT

Drop-off Locations & Times:

Sample drop off times are as follows:

• Blalock 1004: 4 PM for daily processing. Samples may be dropped off after-hours in our fridge near the Ross bridge. The combination for the fridge door is given in your order confirmation. For same day results, samples must be in Blalock 1004 by 9 AM. Additional charges and sample minimums apply.
• CRB1 Rm B02A, in the specifically designated mini-fridge. Pickup will happen after 10 AM.
• WBSB 3rd floor, near the Rangos bridge.  Pickup will happen after 12 noon.
• Bayview Asthma & Allergy building 1st floor hallway, in the designated mini-fridge: Pickup will happen after 12 noon, when the CoreStore is making deliveries. Samples may also be given to CoreStore personnel.
• Homewood: Two locations. Call 410-955-2836 for the combination to unlock the refrigerators.

– UTL: Samples should be in the UTL Rm 384 by 11 AM (Black refrigerator in the break room right across from the main staircase.)

– Croft Hall: Black refrigerator in the hallway outside 141. Samples should be in the refrigerator by 11 AM.

Turnaround Time:

Monday – Thursday: Samples dropped off in the Blalock building by 4 PM or in our remote locations by the appropriate drop off time will be ready by 10 AM the following business day. Samples dropped off after 4 PM will be held for processing the next day.

Friday: Samples dropped off in Blalock by 12:30 PM will have data available on Saturday morning.  Samples dropped off at our remote locations anytime on Friday or in Blalock 1004 after 12:30 PM will enter our regular queue and will be available by 1 PM on Monday.

Same day sequencing service is available at a higher fee per sample, with a minimum of 5 samples. For same day sequencing, samples must be ordered on the  JHU ordering server and physically present in our facility by 9 AM and the data will released by 5 PM.

Failing samples will be rerun on the following business day.

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