Jra3 Protocols For dna extraction From Bone and Tissue Protocols For Preparation of dna extracts For ngs

(1) cleaning of surfaces, utensils and equipment with bleach/UV prior to use.

(2) use of protective clothing to stop tracking of DNA into the lab.

(3) working within a laminar flow hood or glove box to provide a clean space.

(4) including negative extraction and PCR controls alongside samples to improve detection of contaminants.

Extraction Protocol Bone
This protocol can be applied to bone powder of between 10–60mg. PrediCtoR (JRA1 York) should be used as a guide to assess the extent of DNA preservation, and may therefore inform on the appropriate weight of bone powder. It should be noted that the relationship between sample preservation, and the amount of starting material to use is not well characterised, and that while micro-sampling (10mg) is feasible for well preserved materials, it may be insufficient for older/less well preserved samples.
Bone powdering:
There are two main powdering techniques:

(1) pulverization, using a shaking bead in a bone mill.

(2) Directly drilling into the sample.
In both cases prevention of cross-sample contamination should be addressed. The bone powdering area should be separate/enclosed from the PCR set up area. The area and equipment used (shaking beads/containers and drill/drill bits) must be changed/cleaned with bleach/UV prior to use with each new sample to be powdered.

When using the direct drilling approach the drill should be used in short bursts at a slow speed. This is to prevent the drill bit from heating, the heat generated has been found to significantly decrease the DNA yield (Adler et al. 2011).

For every 5 samples extracted include a control (blank) extraction.

Incubate the samples overnight at 56⁰C with gentle rotation.

To separate the supernate from any remaining bone material. Remove samples from the incubator and centrifuge for 5 minutes at 1000 rpm.

Concentrate the DNA using an ultrafiltration membrane. Transfer the supernatant (up to 600L) into a Vivaspin column (500 MWCO). Centrifuge for 2 minutes at maximum speed. A maximum of 120L should remain in the filter column, centrifuge for longer if required.

Per sample set up a 2mL tube containing 600L PB buffer. The buffer contains a high concentration of chaotropic salts that aid adsorption of nucleic acids to silica.

Transfer 120L (maximum) from the Vivaspin filter column into the PB buffer and briefly vortex the solution.

Transfer 720L of the sample solution into a silica-based spin column.

Centrifuge for 1 minute at maximum speed.
Removing impurities:

Transfer the inner column into a new collection tube and add 720L of PE buffer. The buffer contains ethanol to wash away salts and other impurities.

Centrifuge for 1 minute at maximum speed.

Transfer the inner column into a new collection tube.

Centrifuge for 1 minute at maximum speed (dry spin to remove any residual ethanol from the PE buffer).
Elute DNA:

Transfer the inner column into a DNA free tube.

Elution step: add 50L of EB. Wait for 5 minutes.

Centrifuge for 1 minute at maximum speed.

Repeat the elution step and centrifuge for 1 minute at maximum speed.

Transfer the total extract (100L) into a screw top tube.

Add Tween-20 (final concentration 0.05%) to the extract to aid longer-term viability.
Extracts should be stored at -20⁰C.
Extraction Protocol Tissue
Using QIAamp DNA Micro extraction kit (Qiagen).

For every 5 samples extracted include a control (blank) extraction.

Include the optional carrier RNA to increase yield from degraded samples.

At the elution step add 50L of AE buffer and wait for 5 minutes before centrifuging for 1 minute. Then repeat this step.

Transfer the total extract (100L) into a screw top tube.

Add Tween-20 (final concentration 0.05%) to the extract to aid longer-term viability.
Extracts should be stored at -20⁰C.
Library Building Protocol
This protocol can be applied to DNA extracted through either of the aforementioned methods and to PCR products. The protocol is a JRA2 (Mainz laboratory) modified version of the methods described in Meyer and Kircher (2010). Note, there is no fragmentation step due to the (already) fragmented nature of ancient/degraded DNA samples.

For every 5 libraries constructed include a control (blank) library.

A * indicates a PTO bond.

Incubate the reactions in a thermal cycler: 95⁰C for 10 seconds, then a ramp from 95⁰C to 12⁰C at a rate of 0.1⁰C/sec.

Prepare a master mix of the reagents below for the number of reactions required.

All enzymes should remain on ice. Once prepared do not vortex the master mix, use a pipette to combine reagents.

Reagent	Volume (L)	Final conc.
Water	7.12
T4 Ligase Buffer (10x)	7	1x
dNTPs (each 25mM)	0.28	100M each
BSA (10mg/mL)	0.7	0.1mg/mL
T4 PNK (10U/L)	3.5	0.5 U/L
T4 DNA polymerase (5 U/L)	1.4	0.1 U/L
Total	20

Pipette 20L of master mix into a PCR tube and then add 50L of DNA extract. Total per sample 70L.

Prepare a master mix of the reagents below for the number of reactions required.

All enzymes should remain on ice. The master mix can be vortexed, but prior to the addition of the DNA ligase.

Reagent	Volume (L)	Final conc.
Water	14.5
ATP (100mM)	0.5	0.5mM
PEG-4000 (50%)	10	5%
T4 DNA Ligase (5 U/L)	2.5	0.125 U/L
Total	27.5

Add 2.5L of pre-prepared adapter mix (P5 and P7) to each sample. The total volume/sample is 72.5L.

Pipette 27.5L of master mix into the PCR tube containing the combined adapter mix and sample. The total volume/sample is 100L.
Incubate at 22⁰C for 30 minutes.
Purification Step:
Use MiniElute PCR Purification Kit:

Add 200L PB Buffer to samples (total volume/sample is 300L). Transfer to column and spin for 1 minute.

Add 650L PE Buffer into the column and spin for 1 minute.

Dry spin for 1 minute.

Elute using 22L of warm EB Buffer.
Adapter Fill In:
To fill in any gaps or nicks created by the adapter ligation step.

Prepare a master mix of the reagents below for the number of reactions required.

All enzymes should remain on ice.

Reagent	Volume (L)	Final conc.
Water	14.1
ThermoPol Buffer (10x)	4	1x
dNTPs (each 25mM)	0.4	250M each
Bst DNA Polymerase, Large Fragment (8U/L)	1.5	0.3U/L
Total	20

Add 20L of the eluted sample to 20L of the master mix (total volume/sample is 40L) and vortex.

Add 200L PB Buffer to samples. Transfer to column and spin for 1 minute.

Add 650L PE Buffer into the column and spin for 1 minute.

Dry spin for 1 minute.

Elute sample libraries using 33L of warm EB Buffer.

Elute control libraries using 11L of warm EB Buffer.

Prepare a master mix of the reagents below. Prepare 3 reactions per sample library (this reduces the number of PCR duplicates) and 1 reaction per control library.

All enzymes should remain on ice.

Reagent	Volume (L)	Final conc.
Water	29.6
Pfu Turbo Cx Buffer (10x)	5	1x
dNTPs (each 25mM)	0.4	200M each
BSA (10mg/mL)	2	0.4mg/mL
Primer: IS4 (10mM)	1	0.2M
Pfu Turbo Cx Taq. (2.5U/L)	1	0.05U/L
Total	39

Add 3L of index to each purified sample. Add 1L of index to each purified control.

Add 11L of index/sample mix to each reaction tube (3 reaction tubes per sample, 1 reaction tube per control). Each reaction tube should contain a total volume of 50L.

Note: Depending on the samples/research undertaken Pfu Turbo Cx Taq can be replaced with other Taq polymerase. See Dabney and Meyer (2012).

Add 200L PB Buffer to samples (total volume/sample is 250L). Transfer to column and spin for 1 minute.

Add 650L PE Buffer into the column and spin for 1 minute.

Dry spin for 1 minute.