TITLE: Indexing PCR and purification of dsDNA libraries
AUTHORS: Marcel Keller, Christiana L Scheib, Biancamaria Bonucci

[DESCRIPTION]
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:
 


[GUIDELINES]
Please read the general guidelines for working in the Ancient DNA protocol collection – University of Tartu, Institute of Genomics.

[STEPS]
SECTION: PCR
1. In the modern lab, place the PCR strips in the cycler and run the following program:
 
      StepTime [min:sec]Temperature [°C]CyclesPreincubation5:00941Melting0:309415Annealing0:3060Elongation0:3068Hold7:00721Coolinginfinite41
 
SECTION: Purification
3. Turn on the heat block 37 °C for the elution.
SECTION: Purification
6. Label MinElute columns.
Label the 50 ml waste tube.
SECTION: Purification
7. Label tubes:

 


SECTION: Purification
8. Add 500 µL PB buffer (binding buffer) to the MinElute column.

SECTION: Purification
9. Add the (first) PCR reaction (100 µL) and pipette-mix.

SECTION: Purification
10. Spin 13 rpm, 1 min.

SECTION: Purification
11. Discard flowthrough into your waste tube.
SECTION: Purification
2. Add 690 µL µl PE buffer (wash buffer), change tip for every sample.

SECTION: Purification
3. Spin 13 rpm, 1 min .
SECTION: Purification
4. Discard flowthrough into your waste tube.
SECTION: Purification
5. Spin 13 rpm, 1 min (dry spin).
SECTION: Purification
6. Put column in labeled tube.
SECTION: Purification
7. Elute in 35 µL EB buffer (elution buffer), incubate at 37 °C for 10 min.

SECTION: Purification
8. Spin 13 rpm, 2 min.
SECTION: Purification
9. Check that there is liquid in your tube, throw away the column and close your tube.
SECTION: Purification
10. Place in the freezer (-20 °C).
SECTION: Purification
13. Add 500 µL PB buffer (binding buffer) to the MinElute column.
SECTION: Purification
14. Add the second PCR reaction (100 µL) and pipet-mix.
SECTION: Purification
15. Spin 13 rpm, 1 min.
SECTION: Purification
16. Discard flowthrough into your waste tube.
SECTION: Purification
17. Add 690 µL µl PE buffer (wash buffer), change tip for every sample.

SECTION: Purification
18. Spin 13 rpm, 1 min .
SECTION: Purification
19. Discard flowthrough into your waste tube.
SECTION: Purification
20. Spin 13 rpm, 1 min (dry spin).
SECTION: Purification
21. Put column in labeled tube.
SECTION: Purification
22. Elute in 35 µL EB buffer (elution buffer). Change tip for every sample. 
SECTION: Purification
24. Spin 13 rpm, 2 min.
SECTION: Purification
25. Check that there is liquid in your tube, throw away the column and close your tube.
SECTION: Purification
26. Put the tubes into the fridge (if stored for max. 1 day) or freezer (if stored for multiple days).

SECTION: Purification
10. Place in the freezer (-20 °C).

SECTION: Purification
9. Check that there is liquid in your tube, throw away the column and close your tube.
SECTION: Purification
8. Spin 13 rpm, 2 min.
SECTION: Purification
7. Elute in 35 µL EB buffer (elution buffer), incubate at 37 °C for 10 min.

SECTION: Purification
6. Put column in labeled tube.
SECTION: Purification
5. Spin 13 rpm, 1 min (dry spin).
SECTION: Purification
4. Discard flowthrough into your waste tube.
SECTION: Purification
3. Spin 13 rpm, 1 min .
SECTION: Purification
2. Add 690 µL µl PE buffer (wash buffer), change tip for every sample.

SECTION: Purification
1. Discard flowthrough into your waste tube.
SECTION: Purification
8. Spin 13 rpm, 1 min.
SECTION: Purification
7. Add the (first) PCR reaction (100 µL) and pipette-mix.
SECTION: Purification
6. Add 500 µL PB buffer (binding buffer) to the MinElute column. You can use the same tip.
SECTION: Purification
5. Label tubes:
 



SECTION: Purification
4. Label MinElute columns with sample IDs.
SECTION: Purification
3. Turn on the heat block 37 °C for the elution.
SECTION: Purification
9. Add 500 µL PB buffer (binding buffer) to the MinElute column.

SECTION: Purification
10. Add the (first) PCR reaction (100 µL) and pipette-mix.

SECTION: Purification
11. Spin 13 rpm, 1 min.

SECTION: Purification
12. Discard flowthrough into your waste tube.
SECTION: Purification
13. Add 690 µL µl PE buffer (wash buffer), change tip for every sample.

SECTION: Purification
14. Spin 13 rpm, 1 min .
SECTION: Purification
15. Discard flowthrough into your waste tube.
SECTION: Purification
16. Spin 13 rpm, 1 min (dry spin).
SECTION: Purification
17. Put column in labeled tube.
SECTION: Purification
18. Elute in 35 µL EB buffer (elution buffer), incubate at 37 °C for 10 min.

SECTION: Purification
19. Spin 13 rpm, 2 min.
SECTION: Purification
20. Check that there is liquid in your tube, throw away the column and close your tube.
SECTION: Purification
21. Place in the freezer (-20 °C).

2. 
 

SECTION: Purification
7. 
SECTION: Purification
4. Label the 1.5 ml EB tube and aliquot: [# of samples]×35 µl plus 10%.
SECTION: PCR
2. 
SECTION: PCR
3. 
SECTION: Purification
9.1. 
SECTION: Purification
10.1. 
SECTION: Purification
1. 
SECTION: PCR
2. 
SECTION: Purification
12. 
 

SECTION: Purification
23. Incubate at 37 °C for 10 min.
SECTION: Purification
5. Prepare PE (wash) buffer by adding ethanol and aliquoting to 50 ml tubes.