Great turn around time on my 80 exome project!
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The radius is equal to the distance in millimeters between the axis of rotation and the bottom of the gel bed when the plate and the receiver are placed in the plate carrier in the centrifuge bucket.
To achieve RCF = 850 x g: rpm = 27,549/√r
Unincorporated dye terminators co-migrate with sequencing products during electrophoresis. The most common peaks occur roughly 70 bases into the sequencing run, and in cases of severe contamination additional peaks may appear in the vicinity of base 120, 220, or higher. Unincorporated dye terminators can appear in purified products for a variety of reasons.
(a) Unincorporated dye terminators will co-migrate with the sequencing products if the sample is allowed to pass down the side of the well rather than through the gel matrix; always use care when applying the sample drop-wise to the center of the gel matrix.
(b) The plates may have been frozen. Freezing irreversibly damages the integrity of the gel matrix and samples processed on these plates will fail.
(c) Centrifugation conditions are essential to the optimum performance of this product. Make sure that the rpm has been calculated correctly and that the spin times are correctly entered. If conditions are correct, it may be necessary to optimize centrifugation protocol for your specific centrifuge, following recommendations described in answers to 4b and 5b.
The recommended protocol assumes that timing starts when the centrifuge starts spinning. Acceleration and deceleration times are highly variable and on some centrifuges they are controllable and on others, they are not. The recommended protocol is an approximation of the best conditions for using this product. It may be necessary to adjust the spin conditions slightly from the recommended protocol to achieve optimum results.
Spin the plate for an additional minute at the correct g force. Even though this procedure may "save" your sample, it will not be optimal. It will likely have low signal strength and may be contaminated with unincorporated dye terminators. You may need to repeat the sequencing reaction.
(a) The plate may be dehydrated. To avoid this, plates should be stored at 4° C in their original packaging.
(b) Centrifugation conditions are essential to the optimum performance of this product. If the centrifuge is new or different, or you are a first-time user of the product, be aware that not all centrifuges respond the same to any given set of instructions. Acceleration and deceleration rates are often different and this may affect your results. First, check the spin conditions and make sure that the rpm has been calculated correctly and the spin times are correct. If the calculations are correct, then consider that centrifuges with fast acceleration rates are particularly susceptible to this type of problem. Try decreasing the length of the initial spin by 30-60 seconds.