Sanger sequencing at the GRCF

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.

Pricing

Single, pre-mixed template & primer: $6.00
Short PCR products (less than 150bp): $6.25
95 samples in a 96 well plate: $5.75 per sample.
BAC protocol reactions (used on BACs, other large templates or very GC rich templates): $25
Same day service: $30 per sample, minimum of 5 samples

Standard primers will be added by our staff. Please indicate which primers are needed on the order form.


Sample Prep 

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.

Custom Primers

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.


Standard Primers

BGH ReverseTAG AAG GCA CAG TCG AGG
CMV-ForwardCGC AAA TGG GCG GTA GGC GTG
EBV-RPGTG GTT TGT CCA AAC TCA TC
KSCGA GGT CGA CGG TAT CG
M13 ForwardTGT AAA ACG ACG GCC AGT
M13 ReverseCAG GAA ACA GCT ATG ACC
MycGCA TCA ATG CAG AAG CTG ATC TCA
Oligo dT-ATTT TTT TTT TTT TTT TTT TTT TTT TA
Oligo dT-GTTT TTT TTT TTT TTT TTT TTT TTT TG
Oligo dT-CTTT TTT TTT TTT TTT TTT TTT TTT TC
Oligo dT-NTTT TTT TTT TTT TTT TTT TTT TTT TN
pDONOR-FPTAA CGC TAG CAT GGA TCT C
pGEX3GGA GCT GCA TGT GTC AGA GG
pGEX5GGC AAG CCA CGT TTG GTG
SP6GAT TTA GGT GAC ACT ATA G
SKTCT AGA ACT AGT GGA TC
SV40pA-RGAA ATT TGT GAT GCT ATT GC
SV40-promotorTAT TTA TGC AGA GGC CGA GG
T3ATT AAC CCT CAC TAA AGG GA
T7TAA TAC GAC TCA CTA TAG G
T7 TerminatorGCT AGT TAT TGC TCA GCG G
U6 (LKO.1)GAC TAT CAT ATG CTT ACC GT

Drop-Off Locations & Turnaround Time

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.
  • 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

  • CRB1 Rm B02A, in the specifically designated mini-fridge.  Pickup will happen after 10 AM.



Turnaround Time:

Samples dropped of in the Blalock building by 4 PM will be ready by 10 AM the following business day. Samples dropped off after 4 PM will be held for processing the next day.

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.

Data Processing

The  JHU ordering server (Genesifter) automatically processes data after electrophoresis is complete and provides quality assessment for each sample. Data is available for download, viewing, and editing.  Please note that Genesifter is only accessible within the Hopkins environment.  To access Genesifter from a remote location outside the JHMI domain firewall, log in to your JHED account and follow the instructions in the sidebar VPN link. Once connected through Pulse Secure you will be able to access Genesifter using the link below.

Location

Johns Hopkins East Baltimore Medical Campus

600 N. Wolfe Street

Blalock Building, Room 1004

Baltimore, MD 21287 USA

Hours

Monday – Friday:

7:00 am to 4:30 pm

After-hours drop-off is available in the fridge near the Ross Bridge on Blalock 10. See your email confirmation for lock codes.

Contact

Phone

410-955-2836


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