User guide

31
Fragment Library Preparation Using the AB Library Builder
™
System: 5500 Series SOLiD
™
Systems User Guide
Chapter4 Nick-Translate the Library with Optional Amplification
(Optional) Nick-translate and amplify the library
e. Transfer the supernatant containing the amplified library to a new 1.5-mL
LoBind Tube.
10. Proceed to “Quantitate the DNA” on page 33.
(Optional ) Nick-translate and amplify the library
Library amplification is useful to increase the amount of rare or low-input samples and
to enrich targeted sequences. Library amplification can, however, bias the library and
introduce base incorporation errors.
Prepare the
reaction, then nick-
translate and
amplify the library
1. In a new 1.5-mL LoBind Tube, combine for a PCR master mix:
2. Transfer 400 µL of the PCR master mix to each library. Each library is 100 µL in an
elution tube so that the total volume of the mix is 500 µL.
3. Vortex the reaction for 5 seconds, then pulse-spin.
4. Distribute 125-µL aliquots of combined library and PCR master mix between
four, 0.2-mL PCR tubes.
IMPORTANT! The current protocol is optimized for maximum yield from input
DNA. In many cases, library amplification is not needed. Quantitate the library to
assess the need to amplify it. If library amplification is needed, minimize the
number of cycles, based on the amount of starting input DNA. Use minimal
cycling to avoid over-amplification and production of redundant molecules.
5. Determine the number of PCR cycles:
IMPORTANT! Minimize the number of PCR cycles to avoid over-amplification and
redundant molecules. Base the number of cycles on the amount of starting input
DNA.
Component
Volume per
amplification
Master mix for N
libraries
Platinum
®
PCR Amplification Mix 380 µL 380 µL × (1.1 × N)
Library PCR Primer 1, 50 µM 10 µL 10 µL × (1.1 × N)
Library PCR Primer 2, 50 µM 10 µL 10µL×(1.1×N)
Total 400 µL 400 µL × (1.1 × N)
Starting amount of DNA
Number of
cycles
10–100 ng 10 cycles
100 ng–1 µg 6–8 cycles
1–2 µg 4–6 cycles
2–5 µg 3–6 cycles