We aim for a fast turn around time, 24 hours or less if you prepare the samples using the information below. Much of the process is automated, the speed at which we can load and run samples depends on how the samples are presented to us. We need to work within constraints of the computer systems we use to control our instruments. File names and sample size are the most important. If we have to work with individual samples that do not conform, it takes longer for everyone to get their data and the per sample price goes up.
Please deliver your samples in the following manner. Samples should be delivered in 1.5 ml eppendorf tubes. Final sample volumes should be at least 20 microliters if they are lower add Milli-Q water to bring them to volume. The exact volume is not important the sequencers perform an electrophoretic injection however, the liquid handling equipment requires at least a 20 ul sample.
Sample names should be kept as short as possible to reduce the possibility of typographical errors. To conform to our new automated data entry system sample names should begin with two initials then a number. For multiple samples the numbers must be sequential as in the following example. For Tom Jones samples TJ1, TJ2, TJ3, TJ4 etc. You can use a batch format such as SK1 to SK12 or an accumulating series corresponding to your lab database to cover all samples submitted over a period of time such as SK142 to SK154. We have labs using both methods. The sample name is used as the beginning of the data file name. At the end of the sequencing run the computer sorts the files alphabetically if all the samples start with the same letter they sort to the same column of data files expediting sample data distribution and it will ease our transition to an automated data distribution system. Please do not use punctuation marks as we are using PCs. PCs will not accept most punctuation marks for use in file names.
The Molecular Biology Core facility distributes ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kits v3.0 With AmpliTaq DNA polymerase, FS at cost. The kits are available in various sizes to accommodate large and small projects. The MBCore tests each lot before we distribute it. We advise reading the manual for kit before preparing your template and primers. You can download this manual in PDF format. An abstracted version, containing only the essential information necessary to process your sample, appears below. Please refer to the manual for information regarding the preparation of a template for sequencing or visit the Core facility and ask. High quality template and a good primer are essential for obtaining good results. Quantitation of the template is of primary importance. Taking an OD of your sample is not the best way to estimate the amount of DNA in the sample. We recommend running the template on an agarose gel with quantitative DNA standards this will produce the best estimate of the amount of DNA in the preparation. For a successful sequencing reaction the molar ratios must be within the windows specified below for the various types of template.
We are currently recommending "half" reactions, using half the 8 microliters of reaction mix recommended by the manufacturer. The addition of 5X buffer allows for a 20 microliter reaction volume which is the smallest reaction the average thermocycler will handle. If you have a thermocycler capable of 10 ul reaction volumes eliminate the buffer and q.s. to 10 ul. IF YOU DO 10ul REACTIONS YOU MUST ADD 10 ul OF MILLI-Q WATER TO YOUR REACTIONS BEFORE PURIFICATION.
Please pay particular attention to the section on purifying extension products below.
For each reaction, mix the following reagents in a labeled tube of the appropriate size:
|
Reagent |
Amount |
Concentration |
Volume |
|
BigDye Terminator Mix |
4ul |
||
|
Double stranded template |
200-500 ng |
Not to exceed 10.8ul |
|
|
Single stranded template |
50-100 ng |
Not to exceed 10.8ul |
|
|
PCR Product 100-200 bp |
1-3 ng |
Not to exceed 10.8ul |
|
|
PCR Product 200-500 bp |
3-10 ng |
Not to exceed 10.8ul |
|
|
PCR Product 500-1000 bp |
5-20 ng |
Not to exceed 10.8ul |
|
|
PCR Product 1000-2000 bp |
10-40 ng |
Not to exceed 10.8ul |
|
|
PCR Product >2000 bp |
40-100 ng |
Not to exceed 10.8ul |
|
|
cosmid, BAC |
0.5-1.0 ugm |
Not to exceed 10.8ul |
|
|
Primer |
1pmole/ul |
3.2ul |
|
|
5x Buffer*1 |
2ul |
||
|
dH2O |
q.s. |
||
|
Total Volume |
20ul |
*The Core facility supplies the 5X buffer at no cost.
We recommend making a "master mix" containing enough BigDye and 5x buffer in the above proportions then adding 6 ul to each reaction containing DNA, Primer and MillI-Q water in the amounts specified above.
Remember this protocol was developed for use with PE/ABI thermal cyclers. If you have a different make of thermal cycler you may need to modify these conditions. Regardless of the thermocycler that you use we advise heating the reactions to 96 degrees C for 5 minutes before cycling. If using the DNA Thermal Cycler (TCI) or the DNA Thermal Cycler Model 480, overlay the reaction mixture with one drop of light mineral oil (approximately 40 mL).
Cycle Sequencing on the GeneAmp PCR Systems 9600 and 2400
1.) Place the tubes in the thermal cycler, begin thermal cycling as follows:
- Rapid thermal ramp to 96 degrees C
- 96 degrees C for 10 seconds
- Rapid thermal ramp to 50 degrees C
- 50 degrees C for 5 seconds
- Rapid thermal ramp to 60 degrees C
- 60 degrees C for 4 minutes
2.)Repeat for 25 cycles.
3.)Then rapid thermal ramp to 4 degrees C and hold.
4.)Purify extension products.
Note: Condensation is sometimes observed on the walls of the tubes at the end of the reaction. If this is the case, it is recommended that the reaction mixture be centrifuged prior to removal of the unincorporated dye terminators.
Cycle Sequencing on the DNA Thermal Cycler (TCI) and the DNA Thermal Cycler Model 480
1.) Place the tubes in a thermal cycler, and begin thermal cycling as follows:
- Rapid thermal ramp to 96 degrees C
- 96 degrees C for 30 seconds
- Rapid thermal ramp to 50 degrees C
- 50 degrees C for 15 seconds
- Rapid thermal ramp to 60 degrees C
- 60 degrees C for 4 minutes
2.)Repeat for 25 cycles.
3.)Then rapid thermal ramp to 4 degrees C and hold.
4.)Purify extension products.
The purification of the extension products is very important. If the unincorporated dye terminators are not thoroughly removed from the reaction your sequence will be rendered unreadable due to the overwhelming fluorescence of the aggregated dye terminators. There are several acceptable methods for removing the unincorporated dye terminators. The cheapest way to do it is ethanol precipitation, however we have found it to unreliable and do not recommend it. We recommend spin column purification of extension products. Two products are available for this procedure. The Remsen Stock room carries Edge BioSystems columns. These columns are relatively inexpensive and give good results on the average sample. However they are known to give less than a quantitative yield of extension products and have some batch to batch variation. A more quantitative product is available from Princeton Separations. These columns give a better yield of extension products however, they are not as convenient to use and are more expensive. Whichever column you choose be sure to follow the package directions carefully pay particular attention to calculating the G force in the centrifugation step. There are other methods available such as magnetic bead technology and a membrane filtration method however, they are designed for high through put 96 well plate technology and would be of little use for small numbers of samples. If you intend to do full 96 well plate sequencing please visit the core and allow us the assist you in setting it up. There is a discount for a full plate of completed samples but there are some specific guidelines.