FAQJump to Sequencing FAQ
Jump to Synthesis FAQ
- How should I provide my template and primer?
Please provide your pre-mixed sample containing 6 µl of template and 6 µl of primer, at the following concentrations:
PCR products: 3.0 ng/µl/100 bp.
Plasmid DNA: 0.2µg/µl.
BAC/PAC DNA: please contact the lab.
If using the primers available at DAF, please bring 6µl of template in a tube and add 6µl of the appropriate primer when you arrive at our facility. There is no charge for using our primers.
- How do I place a sequencing order?
The JHU ordering server manages all orders and data. To place an order and view your data, you must have a username and password. If you need a JHU Finch server login, please call us at (410) 955-2836 and we will be happy to assist you. Please check our How-To section for detailed help with placing a sequencing request.
Please remember, in order to utilize all of the features of the server, including order notifications and data tracking, each individual in your lab should have their own login.
- Should each person in a lab have their own log-in for the Finch server?
Yes. If you use your own log-in to create your orders, you will receive an e-mail when your data is available. This also provides the lab a correct contact name and number when there is a problem with a request. PIs who have their own log-in can have their accounts set up so that they can manage and access all data for their lab.
- When will my data be ready?
Results for sequencing samples dropped off in Blalock by 1:00 PM will be available by 10 AM the following morning, assuming no machine problems. Samples dropped off by 3:00 PM will be available the following afternoon. Samples dropped off after 3 PM will enter the next day's cycle. You will receive an email when your data is completed.
- Where do I drop off my samples?
Blalock 1005: Open 7 AM - 4:30 PM. There is also an after hours drop-box available for after-hours.
CRB I B02A: Samples dropped off here by 2:00 PM will normally be picked up and processed the same day, making the data available the following afternoon.
Bayview Campus: Drop off in the Core 24/7 on the first floor of the Asthma and Allergy building, in slot F51. Pickup is once daily. Samples dropped off by 11:45 AM will have data available by 10 AM the next morning. Samples dropped off after 11:45 will wait for the next day. First time users will need to email Lee Hilliard or call 410-502-3959 to set up and account. You may also download forms for 24/7 access. If you already have a 24/7 account, you can just login to the system and drop off the sample. Someone is on campus every day from 11:00 AM to 12 PM to help with problems and questions.
- How should I submit my samples?
Tubes: due to storage issues, we will accept only 1.5 ml eppendorf tubes. Plates: 24 samples or more may be submitted on plates with the requirement that all consecutive wells are filled in a top to bottom, left to right format (i.e. A1-H1; A2-H2, etc.).
Labeling: Your tubes need to be labeled with your sample request ID and matching Finch server sample ID.
Note: Any sample that is improperly labeled, unreadable, or any sample sets organized in a way that we feel may increase the risk of error during handling in the DNA Analysis Facility may be rejected. Improperly labeled samples will be returned.
- How much does sequencing cost?
Single template/primer: $8 (pre-mixed). An additional $5 will be charged for samples that are not pre-mixed. Standard primers are available for you at Blalock 1005.
95 samples in a 96-well plate: $7.50 per sample.
Same day service: $20 per sample, with a minimum of 5 samples. Samples must be ordered in the Finch server and physically present in our facility by 9 AM.
- My sequence didn't work. How do I know what is wrong with it?
There are many reasons why a sequencing reaction may fail. Please see our sequencing troubleshooting guide for more info.
- Which synthesis scale should I use?
- How long does it take to receive my oligos?
- Will you deliver my oligos?
- What is the largest oligo I can synthesize?
- I am ordering a lot of oligos at once. Can I upload a spreadsheet?
- Where do I find the codes for special modifications for my oligos (like fluorescent labels)?
- How do I order a specialty probe?
- How do I know which kind of purification to use?
Primers that are being used for PCR or sequencing, and are less than 50 bp, can generally be synthesized on the 0.025 µM scale. Primers which are 50 bp or longer, or which are purified or have a modification, must be synthesized at the 0.05 µM scale or higher. Specialty probes must be ordered by OD scale, and the scale depends on the probe type. Please see the How-To section of the web-site for details regarding specific types of probes.
Standard, small scale synthesis oligo orders placed before 2 PM each day will arrive within 48 hours, barring a synthesis problem. Modified, purified, large scale or long oligos can take up to a week to come. Specialty probes may take up to two weeks to arrive.
If you indicate your building and room number in the delivery section of the order (not your street address), your order will be delivered with the standard Core Store deliveries. Someone must be present in your lab to sign for the oligos. If you would like to pick up your oligos, please select the "Pickup order at lab" option.
Maximum oligo length is 110 bases and must be ordered at 0.05 µM scale or above. Note that long oligos take up to a week to synthesize.
Yes, see the directions on the How-To page.
Click on the blue "i" button at the end of the sequence box. A pop-up window will open. Then select the category of modifier (5', 3' or internal) and find the code for your selected item. Enter that code in the appropriate spot in your sequence. The codes should include the brackets [ ]. If the code for a modifier doesn't exist, please call us and we will add it.
See the instructions for specialty probes on the How-To page.
Oligos being used for PCR, sequencing and other like purposes do not generally need to be purified. It is recommended that oligos longer than 30 base be purified.
Reverse-phase cartridge purification (RP1) is almost equivalent to HPLC and often has greater yields. It is not recommended for oligos greater than 50 bases. This purification method results in products 80-90% pure and is sufficient for most purification needs.
HPLC purification yields a product of 90-97% purity. This is the method of choice for large scale synthesis (1 µmole or greater). It is not recommended for oligos longer than 50 bases.
PAGE has excellent resolution and can achieve 95-99% purity. In most cases full length oligos can be separated from oligos only one base shorter. This is the recommended technique for oligos greater than 50 bases. Yields are generally lower than other methods.
Oligos that have been modified with biotin, fluorescein, 6-FAM, HEX and TET can be purified by any method. HPLC or PAGE is recommended for oligos that have been modified with digoxigenin, the ABI dyes, Texas Red, and any Molecular Probe dye. 5' Phosphorylated oligos cannot be purified with RP1 or HPLC, and must use PAGE.
Each type of specialty probe has its own recommended method of purification. Please see the How-To page for directions.