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Q1: How should my samples be labeled?
A:
Samples should be labeled with your initials and a serial listing. For example, assume your name is Mary Lynch, and you are submitting four samples. Your samples should be labeled ML1through ML4. The next time you submit samples for sequence analysis, begin with ML5 and continue in this fashion. All tubes must be labeled individually.
Q2: When will I get my results?
A:
At this time there is a 24 to 48 hour turn around time for electrophoresis and complete reactions. All samples collected by 8:30 am will be posted by 10:00 am the next day.
Q3: How will I know when my sequencing results are completed?
A:
The facility staff will email you when the results are ready. Make sure we get your correct email address when you enter your samples into the data computer at the DNA Sequencing Facility. You will not be notified in any other manner other than email that your results are ready to be viewed on the internet. Contact the DNA Sequencing Facility staff to have a new account set-up, this includes a username and password unique to each lab.
Q4: When and where do I drop-off my samples?
A:
Samples can be dropped-off Monday through Friday, between 9:00 am and 5:00 pm in HSC T-8, Rm 047B. Also we have two (2) drop boxes located at Life Sciences Building 2nd floor Room 253 and at CMM (Center for Molecular Medicine) 4th floor Room 450. The drop off time at these two locations is between 9:00 am and 5:00 pm.
For electrophoresis only samples: leave your samples dried-down in 0.2ml strip tubes in the gray racks in the refrigerator labeled "electrophoresis only". For complete reactions: leave your samples in 0.2ml strip tubes in the gray racks in the refrigerator labeled "completes only". (See question 6 for more info.)
Q5: How much DNA is required for sequencing?
A:
The recommended quantities of DNA are as follows:
| Template and Primer Quantities |
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| Overview | | If possible, quantitate the amount of purified DNA by measuring the absorbance at 260 nm or by some other method. |
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| Template Quantity | | The table below shows the amount of template to use in a cycle sequencing reaction. |
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| Template | Quantity |
|---|
PCR product:
100-200 bp
200-500 bp
500-1000 bp
1000-2000 bp
>2000 bp |
1-3 ng
3-10 ng
5-20 ng
10-40 ng
40-100 ng |
| Single-stranded | 50-100 ng |
| Double-stranded (Plasmid DNA) | 250-500 ng |
| Cosmid, BAC | 0.5-1.0 µg |
| Bacterial genomic DNA | 2-3 µg |
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| Note: | In general, higher DNA quantities give higher signal intensities, however, if the quantities above are exceeded problem will occur with sequencing. |
| Note: | The template quantities stated above should work with all primers. You may be able to use even less DNA, especially when sequencing with the -21 M13 primer. The amount of PCR product to us in sequencing will also depend on the length and purity of the PCR product. |
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| Template Volume | | Cycle-sequencing reactions are made up in a final volume of 8µl. The volume includes DNA template, primer, and PCR water. If your DNA is not concentrated enough and you need to add more DNA template, then you can compensate for the additional volume by using a more concentrated solution of primer. |
Q6: How should I prepare my samples for sequencing service?
A:
Samples should be in 0.2ml strip tubes. Note that only this type of tube will be accepted.
| Preparing sequencing samples: |
| 1. | Combine the following in a PCR tube: sample DNA (please refer to the above table in Q5 for the amount of DNA to use), 3.2pmol primer/sample, PCR water to 8µl. The total volume of each sample should equal approximately 8µl, you change the volumes of solutions depending on your concentrations of DNA and primer. At this point samples can be brought to the DNA Sequencing Facility for complete service. For electrophoresis only service continue the following protocol. |
| 2. | Add 2µl per sample of the Big Dye Terminator version 3.1. |
| 3. | Run the sample in a ThermalCycler using the Big Dye protocol as follows: |
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Step 1 - Incubate at 96°C for 20 seconds
Step 2 - Incubate at 50°C for 20 seconds
Step 3 - Incubate at 60°C for 4 minutes
Step 4 - Repeat to Step 1 for 39 times
Step 5 - Incubate at 12°C forever
Step 6 - End
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| Note: |
| If preparing samples that include bacterial DNA the sample prep will be the same but the ThermalCycler protocol will be slightly different, as follows: |
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Step 1 - Incubate at 95°C for 20 seconds
Step 2 - Incubate at 45°C for 20 seconds
Step 3 - Incubate at 60°C for 4 minutes
Step 4 - Repeat to Step 1 for 39 times
Step 5 - Incubate at 12°C forever
Step 6 - End
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| Cleaning the Completes Using Gel Columns: |
| 1. | Centrifuge the Performa DTR Gel Filtration Cartridges in a centrifuge at 3000rpm for 2 minutes. |
| 2. | Discard the original 1.5ml tube under the centrifuged gel column-this 1.5ml tube should have water in the bottom from the gel column. |
| 3. | Place a clean 1.5ml tube under the centrifuged gel columns, pipette 12µl of each sample (those taken out of the PCR machine) into the corresponding tube, and centrifuge for 2 minutes at 3000rpm. |
| 4. | Take all samples after centrifuging and discard gel column and transfer sample from the1.5ml to a 0.2ml strip tube and place tube in vacuum with the lid open for 30 minutes or until the samples are completely dry. Remember to leave the lids open on the tubes. (This step evaporates all water that may be in the sample and only leaves the wanted DNA.) If you do not have adaptors for 0.2ml tubes you can use 1.5ml tubes as adaptors in the rotors. |
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| Cleaning the Completes using ethanol precipitation |
| Add the following reagents to the PCR product as follows: |
| 1. | NH4OAc (7.5M) | | 1µl |
| 2. | Ethanol (100%) | | 100µl of 100% |
| 3. | Initial Spin | | 15 min. @ 3500rpm-maximum speed |
| 4. | Discard Supernatant - be careful not to discard the pellet |
| 5. | Final ethanol wash (70%) | | 200µl of 70% |
| 6. | Final spin | | 7.5 min. @ 3500rpm-maximum speed |
| 7. | Discard Supernatant - be careful not to discard the pellet |
| 8. | Dry samples in a vacuum, preferably using a rotor. (This step evaporates all water and/or ethanol that may be in the sample and only leaves the wanted DNA.) |
Q7: What is the cost of running a sample?
A:
Our current price is $13.88 per sample for SUNY Stony Brook users for the electrophoresis
only. For complete service (PCR reaction and electrophoresis), the
cost is $6.25 for SUNY Stony Brook users. Non-SUNY Stony Brook users need to submit a check
made payable to "IFR 910076 Physiology and Biophysics" with each
request for sequencing.
Q8: Where can I get the BigDye Kit?
A:
The BigDye Kit can be purchased from Applied Biosystems or from the DNA Sequencing Facility.
Applied Biosystems
850 Lincoln Centre Drive
Foster City, CA 94404
1-(800) 345-5224
BigDye Terminator version 3.1 (version 3.0 discontinued June 2003)
| P/N | KIT # | REACTIONS |
|---|
| 4337454 | Ready Reaction | 24 |
| 4337455 | Ready Reaction | 100 |
| 4337456 | Ready Reaction | 1000 |
Additionally, the BigDye kit can be purchased directly from us in
10 reaction and 100 reaction aliquots. The cost is $10.35/per 8ul per reaction for internal users.
Q9: Can I get my results saved on floppy/zip disk?
A:
No, from now on your results can only be obtained through the internet. No more floppy or zip disks will be accepted. If you do not have an internet account with us, please let us know and we'll open one for your lab.
Q10: Why did my samples give weak or no readable sequence?
A:
The most common causes for sequencing failure:
| 1. | | Poor quality of DNA. How was the DNA purified? |
| | (a) Miniplasmid DNA must be RNase and Protease A treated before phenolextraction and ethanol precipitation.
(b) Analytical preparations of DNA are preferable, such as CsCl gradient or one of the commercially available kits: Qiagen, Promega Maxi Kit, etc.
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| 2. | | Too high a concentration of EDTA in your reactions, which inhibits Polymerase activity. This usually happens when large volumes of dilute DNA in TE are used. Remedy: prepare DNA in TE 0.1 or concentrate DNA by ethanol precipitation. |
| 3. | | Concentration of primer. Check a 260nm of stock primer solutions and prepare new working solutions. Primer in dilute solutions tend to stick to the sides of tubes, so vortex thoroughly before pipetting. |
| 4. | | Faulty "clean-up" of reaction. This maybe due to loss of DNA during ethanol precipitation or spin column procedure. Follow protocols outlined in kit. |
Q11: Will I get charged if my samples gave little or no readable sequence?
A:
Yes, unless the fault is ours; e.g. computer collection
glitch or other hardware problem. Several control samples are
processed on every run and the results of these standards are posted
with your results in the bins outside the lab for you to consult. If
you would like to obtain a copy of our control results, please ask one
of the faculty members and we would be happy to provide you with a
copy. In the event of a facility problem, we will re-run your
sample(s) free of charge.
Q12: How can I get in touch with the facility?
A:
E-mail the facility at: dna@osa.sunysb.edu or Call the DNA Sequencing Facility at 631-444-6849. If no one is available, leave a voicemail and someone will return your call as soon as possible.
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