PCR Clean-up

Exosap-IT

Keep Exosap on ice!
For 2-reaction template volume (if the goal is two-directional sequencing)

1. Mix:

• 0.5uL water
• 2uL template (assuming concentration about 20ng/uL)
• 1uL Exosap

2) Run ExoSap program on thermocycler (37C for 15 min, and 80C for 15 min)

3) Aliquote 1.75 uL of product in sequencing wells

Gel extraction and clean-up

Our choice: E.Z.N.A. Gel Extraction Kit, Omega Bio-tek

• 1) Cut out gel slice, put in a tube, and weigh it (actual weight minus one empty tube (0.90 g))
• 2) Add equal volume of Binding Buffer (x g gel = xml Buffer)
• 3) Incubate at 55-60 C for 7 min (or until gel dissolves)
• 4) Add 700uL to spin column, centrifuge at 10,000g for 1 min
• (5) Keep adding 700uL of solution to the spin column and spinning until all added)
• 6) Add 300uL of fresh Binding Bufferto the column, spin at 10,000g for 1 min
• 7) Add 700uL of ‘Wash Buffer, centrifuge at 10,000g for 1 min, discard flow-through
• 8) Same thing again: add 700uL of Wash Buffer, centrifuge, discard liquid
• 9) Discard liquid, centrifuge the empty column for 2 min at max speed
• 10) Put column in clean tube, add 30uL of Elution Buffer
• 11) Let soak for 1 minute, then centrifuge at max speed for 1 min
• (12) (If maximum yield is more important than concentration, add another 30uL of Elution Buffer, centrifuge at max speed.)

Notes: When DNA yield is important, use fresh running TAE buffer. Old buffers have high pH and decrease yield.

If Buffer turns orange or red during incubation, pH is too high!

Useful overview of available kits here.

Genewiz folks recommend Qiagen kit, but that is expensive and people report low yields. If you need to load and separate the whole PCR reaction, make a thick gel.

MOBIO UltraClean PCR Cleanup

Protocol modified by Fierer Lab

• 1. Shake to mix the SpinBind before use. Add 5 volumes of SpinBindto your PCR reaction. (ex. 500uL to 100uL of PCR reaction)
• 2. Mix well by pipetting. If an oil overlay was used, you will now have two layers. The top layer is oil.
• 3. Transfer PCR/SpinBind mixture to a Spin Filtercolumn tube, while avoiding the transfer of oil.
• 4. Centrifuge 10-30 seconds at a minimum 10,000 x g~ 13,000rpm in a tabletop microcentrifuge.
• 5. Remove the Spin Filter basket and discard liquid flow through by decanting then replace basket back in to the same tube.
• 6. Add 300 ul SpinClean Buffer to the Spin Filter.
• 7. Centrifuge 10-30 seconds at a min of 10,000 x g
• 8. Remove the Spin Filter basket and discard liquid flow through by decanting then replace basket back in to the same tube.
• 9. Centrifuge 90 seconds at a min of 10,000 x g
• 10. Transfer Spin Filter to a clean 2ml Collection Tube
• 11. Add 50uL Elution Buffer (10mM Tris) solution provided or sterile water directly onto the center of the white Spin Filter membrane. The choice of Tris or water will not affect yield. DNA is more stable for storage in Tris. Let sit on filter for 5 minutes.
• 12. Centrifuge for 90 seconds at a min of 10,000 x g
• 13. Discard Spin Filter basket from the inside of the Spin Filter. Purified DNA is now in the 2mL Collection tube. The DNA will be free of all reaction components such as primer or linkers, enzyme , salt and dNTPs. Store DNA at -20 degrees Celsius. DNA is now ready to use.