Indexing PCR and purification of dsDNA libraries
Marcel Keller, Christiana L Scheib, Biancamaria Bonucci
Abstract
Protocol for the indexing PCR and purification of dsDNA libraries, optimized for ultra-short ancient DNA molecules, modified from Meyer & Kircher (2010) Cold Spring Harb. Protoc. (doi: 10.1101/pdb.prot5448).
Before start
Previous step:
This protocol follows the library preparation protocols.
Following step:
After the purification, the libraries are ready for quality control.
Equipment and consumables:
| A | B |
|---|---|
| Number | Equipment and consumables |
| 1 | 0.2 ml tube rack |
| 1 | 1.5 ml tube rack |
| 1 | 50 ml Falcon rack |
| 100 µl filter tips | |
| 200 µl filter tips | |
| 1000 µl filter tips | |
| [# of samples]+1 | 1.5 ml tubes |
| [# of samples] | MinElute columns |
| 1 | 50 ml Falcon (waste) |
[# of samples] includes the blank(s).
Steps
PCR
In the modern lab, place the PCR strips in the cycler and run the following program:
| A | B | C | D |
|---|---|---|---|
| Step | Time [min:sec] | Temperature [°C] | Cycles |
| Preincubation | 5:00 | 94 | 1 |
| Denaturation | 0:30 | 94 | 15 |
| Annealing | 0:30 | 60 | |
| Elongation | 0:30 | 68 | |
| Final elongation | 7:00 | 72 | 1 |
| Hold | infinite | 4 | 1 |
Purification
Turn on the heat block 37°C for the elution.
Label the 1.5 ml EB tube and aliquot: [# of samples]×35 µl plus 10%.
Prepare PE (wash) buffer by adding ethanol and aliquoting to 50 ml tubes.
Label MinElute columns.
Label the 50 ml waste tube.
Label tubes:
| A | B | C |
|---|---|---|
| Top | Project ID | PROJ |
| Library ID | ABC001A 1 SG1 | |
| indices | NEB1 (single) i701 / i501 (double) | |
| Side | Project ID | PROJ |
| Library ID | ABC001A 1 SG1 | |
| indices | NEB1 (single) i701 / i501 (double) | |
| date | 01.01.2021 | |
| initials | XY |
Add 500µL PB buffer (binding buffer) to the MinElute column.
Add the (first) PCR reaction (100µL) and pipette-mix.
Spin 13rpm.
Discard flowthrough into your waste tube.
Add 500µL PB buffer (binding buffer) to the MinElute column.
Add the second PCR reaction (100µL) and pipet-mix.
Spin 13rpm.
Discard flowthrough into your waste tube.
Add 690µL µl PE buffer (wash buffer), change tip for every sample.
Spin 13rpm .
Discard flowthrough into your waste tube.
Spin 13rpm (dry spin).
Put column in labeled tube.
Elute in 35µL EB buffer (elution buffer). Change tip for every sample.
Incubate at 37°C for 0h 10m 0s.
Spin 13rpm.
Check that there is liquid in your tube, throw away the column and close your tube.
Put the tubes into the fridge (if stored for max. 1 day) or freezer (if stored for multiple days).
