Reaction Dilution when you are performing your own cycle sequencing reactions;
| |
Reg. 1/4
|
1/8 rxn
|
1/16 rxn
|
| Total Volume |
5
|
10
|
10
|
| BigDye |
2
|
1
|
0.5
|
| 5X buffer |
0
|
1.5
|
1.75
|
| DNA |
2
|
2
|
2
|
| Primer |
1
|
1
|
1
|
|
H2O
|
0
|
4.5
|
4.75
|
Notes:
If DNA or primer is not concentrated enough, add more DNA and less water.
Recommended amounts per cycle sequencing reaction:
| Primer |
3.2 pmoles |
| plasmid DNA |
300 ng |
| DNA from PCR |
3 ng for products under 200 bp
10 ng for products under 500 bp
20 ng for products under 1,000 bp
40 ng for products under 2,000 bp |
Please note that DNA sequencing can work from PCR amplification and may be more effective than plasmid purification. The PCR product will still need to purified to remove primers and dNTPs. If you are submitting plasmid samples for cycle sequencing, please submit at least 4 microliters containing 100 ng/μl plasmid, and 1.2 pmole/μl primer. We will then take 3 μl of this mix to add to our cycle sequencing mix. If you are submitting sample and primer separately, we would like to add 2 μl of template and 1 μl of primer containing the above suggested concentrations of template and primer.
Submitting samples for Parallab 350 Processing
Plates containing 95 samples can be submitted to the Parallab 350 workstation. We need a minimum of 4 such submissions for cost-effective usage of BigDye on this machine (we distribute 2 μl of BigDye across an entire plate and can't afford to loose much to evaporation). The following is a format that works; One plate with a set of 95 primers at 12.5 pmoles primer/μl along with four plates of 95 templates each (we ask that you leave the H12 well empty for our control). Please note that any one well across these four plates of templates will be using the same primer for sequencing. There are other sample/primer combinations that will work when submitting to this workstation. The machine will mix 190 nl of BigDye (this is part of the charge you will be purchasing when using this machine) with 100 nl of primer and 210 nl of template (or 310 nl of primer and template premixed in a well). The machine performs the cycle sequencing and then cleans the reaction products for loading on the ABI 3730xl DNA Analyzer. This machine has 96 capillaries and they do not work independent of each other. Please call the DNASC at 422-3647 if you would like more information.
