TITLE: DOH Workshop Part 2: Promega Pronex protocol
AUTHORS: Vesa Qarkaxhija, Bryan Wee, Natalie Ring

[DESCRIPTION]
This protocol is to purify extracted dsDNA, removing contaminants (e.g., buffers, proteins, salts, etc.) and low molecular weight DNA (e.g., dsDNA adapters, ssDNA oligonucleotides and nucleotides).

[BEFORE_START]
Ensure that the Pronex Wash Buffer is prepared according to instructions on them (i.e.adding appropriate amount of Ethanol).

[GUIDELINES]
Important points throughout protocol:
 

 

 


Troubleshooting:

My DNA yield is low, do I have to redo the extraction?
There are a few things you can do to increase yield before having to redo the extraction. Try these solutions:
Try re-eluting the DNA from the remaining Pronex beads from step 16 of the Pronex protocol. Repeat steps 14 –16.
Repeat Pronex protocol.
Try re-eluting the DNA from the remaining MagAttract Suspension G beads from step 32 of the DNA extraction protocol. Repeat steps 30, and re-elute by placing the tube onto the thermomixer and incubate at 21°C for 6 min at 1400 rpm.

[STEPS]
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1. Resuspend the Pronex beads by vortexing for 10 sor longer.


 


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2. Into your extracted DNA tube pipette 80 µLof Pronex beads and mix into the sample by slowly pipetting 10 times. 

 

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3. Leave at Room temperature for 10 min.

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4. Place the sample on a magnetic rack for2 min (or longer if necessary) until the solution becomes clear and the beads form a pellet.

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5. While leaving tubes on the magnet, carefully remove and discard supernatant without disturbing the beads using a P1000.
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6. While the tube is on the magnetic rack, add 200 µLof Pronex wash buffer without flushing directly onto the pellet. If 200 µLis not enough to submerge the pellet, use more wash buffer. 

 

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7. Allow to incubate for 30 s-1 min .

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8. While leaving tubes on the magnet, carefully remove and discard wash buffer without disturbing the beads using a P1000.
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9. Keeping the tube on the magnetic stand, carefully add 200 µL of Pronex Wash Buffer without flushing directly onto the pellet. If 200 µL is not enough to submerge the pellet, use more wash buffer.

 

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10. Allow to incubate for 30 s-1 min .

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11. While leaving tubes on the magnet, carefully remove and discard wash buffer without disturbing the beads using a P1000.
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12. Allow the sample to air dry with lids open for 2 min-5 minwatching it until the pellet is no longer shiny.

 

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13. Remove the sample from the magnetic stand.
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14. Remove the tube from the magnetic rack and add 32 µL of nuclease free water. Resuspend the beads by slowly pipetting or stirring with the pipette tip. 

 


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15. Leave for5 min at Room temperature to elute the DNA.

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16. Pellet beads on magnet for 1 min until solution becomes clear and slowly pipette DNA eluate into a new LoBind tube. 

 


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17. Quantify 1 µL of DNA elute using the Qubit™ dsDNA HS Assay Kit or QuantiFluor® ONE dsDNA System.