Apr 05, 2023
Indexing PCR and purification of dsDNA libraries
- 1Institute of Genomics, University of Tartu;
- 2Institute of Forensic Medicine, University of Bern;
- 3University of Cambridge
Protocol Citation: Marcel Keller, Christiana L Scheib, Biancamaria Bonucci 2023. Indexing PCR and purification of dsDNA libraries. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzboy4vx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 31, 2023
Last Modified: April 05, 2023
Protocol Integer ID: 76150
Keywords: ancient DNA, aDNA, archeogenetics, archaeogenetics, paleogenetics, palaeogenetics, library preparation
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).
Guidelines
Please read the general guidelines for working in the Ancient DNA protocol collection – University of Tartu, Institute of Genomics.
Materials
Reagents:
| A | B | C | D | E | F | G | |
| Step | Reagents | Conc. | Unit | Manufacturer | Kit/full description | Product number | |
| Purification | PB Buffer | N/A | N/A | Qiagen | MinElute PCR Purification Kit | 19066 | |
| Purification | PE Buffer | N/A | N/A | Qiagen | MinElute PCR Purification Kit | 19065 | |
| Purification | EB Buffer | N/A | N/A | Qiagen | MinElute PCR Purification Kit | 28006 |
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) |
Lab equipment:
Dead Air Hood
Centrifuge (1.5/2 ml)
Heat block
Mini table centrifuge/vortexer
Other consumables:
DNA ExitusPlus
Paper towels
Safety warnings
Reagents
DNA ExitusPlus
H319 Causes serious eye irritation.
Guanidinium hydrochloride (GuHCl ) (in PB buffer of Qiagen MinElute kit)
- H302 Harmful if swallowed.
- H332 Harmful if inhaled.
- H315 Causes skin irritation.
- H319 Causes serious eye irritation.
Ethanol
- H225 Highly flammable liquid and vapor.
- H319 Causes serious eye irritation.
Equipment
UV radiation
- UV radiation can damage eyes and can be carcinogenic in contact with skin. Do not look directly at unshielded UV radiation. Do not expose unprotected skin to UV radiation.
- UV emitters generate ozone during operation. Use only in ventilated rooms.
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).
PCR
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 |
Note
Continue immediately with the purification or stop here and purify later.
To continue with purification, take MinElute columns out of the fridge while the PCR is running.
To stop and purify later, put the strips into the fridge (if stored for max. 1 day) or freezer (if stored for multiple days) after the PCR is done. For the purification, take the MinElute columns out of the fridge in time so they can reach room temperature until PB buffer and libraries are added.
Purification
Purification
2m
2m
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 500 µL PB buffer (binding buffer) to the MinElute column.
Add the (first) PCR reaction (100 µL ) and pipette-mix.
Spin 13 rpm, 00:01:00 .
1m
Discard flowthrough into your waste tube.
Note
Steps 12 to 14 only apply if you have 2x split the PCR reaction.
Add 500 µL PB buffer (binding buffer) to the MinElute column.
Add the second PCR reaction (100 µL ) and pipet-mix.
Spin 13 rpm, 00:01:00 .
1m
Discard flowthrough into your waste tube.
Add 690 µL µl PE buffer (wash buffer), change tip for every sample.
Spin 13 rpm, 00:01:00 .
Discard flowthrough into your waste tube.
Spin 13 rpm, 00:01:00 (dry spin).
Put column in labeled tube.
Elute in 35 µL EB buffer (elution buffer). Change tip for every sample.
Incubate at 37 °C for 00:10:00 .
10m
Spin 13 rpm, 00:02:00 .
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).