SequalPrep™ Normalization Plate Kit, 96-well

Nested PCR requires two separate amplifications, with the first one using one set of PCR primers and the second one using internal, "nested" primers and 1% or less of the first PCR reaction as a template. Nested PCR is used when the target is present in low abundance or when non-specific PCR products are being produced along with the specific product. Semi-nested PCR is used when there is only enough sequence information to make a primer internal to one end of the primary PCR product such as in RACE (rapid amplification of cDNA ends).

Yes, you can sequence PCR products. This is an excellent way to confirm the sequence of an insert when plasmid sequencing produces an unexpected sequence. The target DNA is amplified with a single set of primers and then sequenced using the same primers (although not all PCR primers work well as sequencing primers). Plasmid DNA can be PCR amplified using a one set of primers and the resulting product can then be purified using a column and then sequenced using the same primers. You can even get more specificity by using a sequencing primer that binds internally to one of the PCR primers.

No, they are not the same. The MicroAmp Optical 96-Well Reaction Plate is an empty optical plate that is used for sample processing (i.e. amplification using the thermal cycler or sample loading onto a capillary instrument). The MicroAmp plate cannot be used for performing normalization as it is not coated using the ChargeSwitch technology.

The SequalPrep Normalization Plate Kit uses ChargeSwitch technology. The ChargeSwitch chemistry is coated on the sides of the plate at the lower portion of the well. When the sample is in the well, a finite amount of sample (approx. 20 ng) is bound to the sides of the well and the rest of the sample stays in solution.
Note: For successful normalization, we recommend using at least 250 ng of PCR product per well
Here are some additional notes to consider when using this product:
- Add the correct amount of Binding Buffer to the PCR sample prior to binding.
- Wash with Wash Buffer and make sure to remove all of the residual Wash Buffer by tapping the plates upside down on paper towel and leaving to drain for 5 mins.
- Elute with Elution Buffer (NOT water).
- Pipetting up and down or gently vortexing followed by centrifugation is the best way to mix.
- Make sure the fluid is at the bottom of the well to get maximum binding, which is where the DNA is captured and eluted.
- The expected amount of DNA after elution is 25 ng with an average range of 2-3 fold variation.

If these suggestions do not resolve the issue, please contact Tech Support at techsupport@thermofisher.com.

We do not recommend incubating for longer than 60 minutes. Maximum binding is achieved within 60 minutes, so extending the binding step will not increase binding. 250 ng of PCR product input is enough to saturate the binding capacity for the plate.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.