SequalPrep™ Long PCR Kit with dNTPs

Does the SequalPrep™ Long PCR Kit replace reagents currently in the Next Generation Sequencing process flow?
No, the SequalPrep™ Long PCR Kit is used to enrich the genome for areas of interest before fragmenting, polishing, adaptor ligation, and amplification in any of the Next Generation Sequencing workflows. The SequalPrep™ Long PCR Kit is used to increase the concentration of the region of interest, up to several megabases in size, with the most robust amplification success rate for amplicons up to 20 kb.

Is the SequalPrep™ Long PCR Kit good for any applications outside of Next Generation Sequencing sample enrichment?
Yes, the SequalPrep™ Long PCR Kit is an excellent long range PCR kit for amplicons up to 20 kb. Individuals have successfully used the kit for additional applications such as genotyping as part of the Knockout Mouse Project.

What is the PCR enzyme used in the SequalPrep™ Long PCR Kit?
The SequalPrep™ Long Polymerase was selected to provide successful amplification for long amplicons with sufficiently high fidelity. In addition, the enzyme mix has an automated hot-start to increase specificity of the reaction. The novel combination of technologies in the SequalPrep™ Long PCR Kit, including the SequalPrep™ Long Polymerase, ensures maximum success for long amplicons.

What is the largest DNA fragment the SequalPrep™ Long PCR Kit can amplify?
The SequalPrep™ Long PCR Kit has been used to amplify amplicons from 5 to 20 kb with a very high success rate. In our hands, depending on template preparation, primer design, and enhancer, templates have been amplified up to 22 kb. In general, Invitrogen scientists get the best amplification success rates when amplifying amplicons between 10 and 15 kb.

What reaction volume do you recommend?
20 μl.

What amount of input material do you recommend?
Most data from Invitrogen R&D scientists has been generated with 50 ng template DNA. Due to the high yields generated by the reaction, some researchers have reported reducing the input material to 10 ng. The general recommendation is 5–200 ng of human genomic DNA, and 20–80 ng of mouse embryonic stem cells DNA.

How many units are used in a typical reaction?
We recommend 1.8 units to a reaction.

Do you have primers designed that are optimized to work with this kit?
Not yet, but we are looking for feedback on what researchers would find useful for their applications.

The kit comes with several Enhancers. What are they and how are they used?
The composition of the Enhancers is proprietary, but they were developed to provide maximum success rates in long PCR. We recommend setting up the initial reactions with 0.5X Enhancer A. For any unsuccessful amplicons, we recommend testing 1.0X Enhancer A, 0.5X Enhancer B, and 1.0X Enhancer B (all provided in the kit).

What organisms have been tested with the kit?
Human and mouse genomic DNA.

What is the fidelity of the SequalPrep™ Long PCR Kit.
The SequalPrep™ Long PCR Kit demonstrates fidelity 3–4 times greater than recombinant Taq, similar to Invitrogen’s Platinum Taq DNA Polymerase High Fidelity Enzyme (by rpsL assay). The SequalPrep™ Long PCR Polymerase was compared to Recombinant Taq DNA Polymerase (Invitrogen), Platinum® Taq DNA Polymerase High Fidelity, and Expand Long Template PCR System (Roche).

Relative fidelity:
Recombinant Taq DNA Polymerase (Invitrogen) = 1
Platinum® Taq DNA Polymerase High Fidelity (Invitrogen) =3.3
Expand Long Template PCR System (Roche) = 3.6
SequalPrep™ Long PCR Kit w/Enhancer A (Invitrogen) = 3.6
SequalPrep™ Long PCR Kit w/Enhancer B (Invitrogen) = 4.4

SequalPrep™ Normalization Plate (96) Kit

What is the largest size amplicon tested for normalization on the SequalPrep™ Normalization Plate (96) Kit?
We have eluted (with high efficiency) templates that were 15 kb and 20 kb in length with the SequalPrep™ Normalization Plate.

What are the DNA yields obtained after elution?
For input amounts of 250 ng or more, the plate elutes an average of 25 ng total DNA. To maintain a consistent concentration across the plate, we recommend inputting amplicons with approximately the same average size.

What is the typical input amount of DNA for the SequalPrep™ Normalization Plate?
Minimum input amounts are usually 250 ng of DNA (typically anywhere between 5–25μ of the PCR reaction) to purify 25 ng DNA per well.

What equipment do I need on my robot to automate the SequalPrep™ Normalization Plate?
None. The SequalPrep™ Normalization Plate requires no special equipment including magnets, shakers, heating, or vacuum.

What if I need extra wash buffer?
SequalPrep™ Plate Wash Buffer is available separately from Invitrogen (Cat. no. A10510-03).

Can I normalize different sized fragments with the SequalPrep™ Normalization Plate?
The SequalPrep™ Normalization Plate primarily operates based on mass, therefore it is best to normalize amplicons of approximately equivalent sizes.