Library Prep Steps- DNA Flashcards
Step 1
- DNA Extraction:
Goal: Isolate the genomic DNA from cells or tissues.
This is typically done using chemical, mechanical, or enzymatic methods to break open the cells and extract the DNA.
Step 2
DNA Fragmentation:
Goal: Break the DNA into smaller fragments (usually 200–800 base pairs in length) to make it suitable for sequencing.
Fragmentation can be achieved using various methods, such as mechanical shearing (e.g., sonication, nebulization) or enzymatic digestion (e.g., using restriction enzymes or transposase).
step 3
End Repair and A-Tailing:
Goal: Prepare the fragmented DNA for adapter ligation.
Fragmented DNA ends may not be in the ideal format for adapter ligation, so “end repair” is done to generate blunt ends or compatible ends.
After repairing the ends, an adenine (A) is added to the 3’ ends of the fragments (A-tailing), which helps ligate the adapters that have a complementary thymine (T) overhang.
Step 4
Adapter Ligation:
Goal: Attach sequencing adapters to the DNA fragments.
Short DNA adapters are ligated to both ends of the fragmented DNA. These adapters are necessary for the DNA to bind to the sequencing platform and serve as primer binding sites during sequencing.
Step 5
Amplification (PCR):
Goal: Enrich the DNA library and ensure there is enough material for sequencing.
The adapter-ligated DNA fragments are amplified using PCR with primers that bind to the adapters. This step increases the number of fragments to make them detectable by the sequencer.
Step 6
Size Selection (optional):
Goal: Select fragments of a specific size range.
The library is often size-selected to ensure that only fragments of the desired size range (e.g., 200–500 bp) are included. This can be done using gel electrophoresis or a bead-based method.
Step 7
Quality Control:
Goal: Assess the quality and quantity of the library.
Methods such as gel electrophoresis, qPCR, or bioanalyzer assays are used to check the library’s quality, size distribution, and concentration.
