1. What is the smallest amount of DNA I can put into TruePrime® reaction?
Typically, the minimum amount of DNA for successful WGA or RCA with TruePrime® is 1 pg. However, amplification is dependent upon the quality of input DNA. Optimal amplification is achieved with 1 ng or more of DNA template.
2. Which is the minimal DNA fragment size for TruePrime® Whole Genome Amplification?
Overall, an average fragment size of 2 kb in a DNA sample is the lower limit. Additionally, highly degraded samples tend not to be amplified evenly across the genome.
3. Can the TruePrime® WGA enzymes work with labelled dNTPs?
Yes, we have tested the addition of different concentrations of FITC-, Cy3- and Cy5-labelled dNTPs, achieving an efficient DNA labelling during the amplification. Nevertheless, the reaction conditions will need to be optimized for each labelled dNTP.
4. What is the maximum number of cells that can be used as input for the TruePrime® Single Cell WGA kit?
We have not detected any negative or inhibitory effect using up to 100 mammalian cells as input.
5. Is TruePrime® Single Cell WGA compatible with all single cell isolation procedures?
TruePrime® Single Cell WGA has been tested using different cell lines and types isolated by different methods (serial dilution, manual picking, FACS, micromanipulation, laser capture microdissection and microfluidics). All of them were compatible with TruePrime® amplification.
6. Is TruePrime® WGA suitable for the amplification of single-stranded DNA like the one obtained after reverse transcription of RNA?
Yes, TruePrime® WGA can amplify ssDNA. The ssDNA molecules should have an average fragment size of 2 kb as the lower limit. DNA molecules below 2 kb can be amplified using the TruePrime® apoptotic cell-free DNA amplification kit.
7. Is there any TruePrime® kit suitable for the amplification of short DNA molecules?
Short DNA molecules ( < 2 kb) are not efficiently amplified by any MDA method. However, the TruePrime® apoptotic cell-free DNA amplification kit has solved this issue by adding an adaptor to each DNA end, which allows the efficient amplification of short DNA molecules following the rolling circle amplification method. See the kit handbook for further details.
8. When to select TruePrime® Single Cell WGA and TruePrime® WGA kit?
If DNA already purified select TruePrime® WGA and select TruePrime® single cell WGA kit if DNA not purified.