Sigma Extract-N-Amp

Spin-down protocol

Keep BSA and the Ex-n-A solution cold, on ice or in a pre-frozen rack.

With BSA as a binding agent

• Less than 10uL of the original sample (or a very small chunk)
• 20uL of Ex-N-A Extraction Solution per sample
• (If you need to crush a beetle part, do the above in a 1.5ml tube and transfer the whole solution to a PCR tube)
• run protocol “Ex-n-Amp” on thermocycler (96C for 10 minutes)
• add 20uL of 3% BSA (to neutralize extra stuff)
  •  shake thoroughly
• spin it down, remove the upper half (20uL) as your final sample
• use the 0.5-1.0uL of this supernatant for PCR

BSA from Fermentas, #00066587, 20mg/mL =~2%

NOTE: This method differs from the manufacturer’s protocol, since their Dilution Solution inhibits PCR.

PowerLyzer

PowerLyzer Ultra Clean kit from MoBio, catalog # 12255-50

Prepare:

12 samples per one round

Ice bucket, or pre-frozen tray in a fridge

Tube heater (dry bath) at 65C

Number all your tubes on top (four per sample, Bead tubes, Spin Filters, and the final PCR tubes)

Steps

1. Spin down sample for 1 min at max speed
2. Take 50uL of sediment and add it to bead tubes (with MicroBead solution and MD1)
3. Heat at 65C for 10 min
4. While waiting, pre-aliquot solutions into tubes:

1 beads + 300uL MicroBead + 50uL MD1

2 100uL MD2

3 900uL: MD3

5. Shake on MoBio vortex adaptor at full speed for 2 minutes

6. Continue according to MoBio manual

…

19. Transfer yield into labeled 250uL PCR tubes. This is our final sample.

Alternative strategies for small volumes of DNA

• Direct PCR may work, but not if lots of DNA is present. For direct PCR add 10 – 15 minutes at 95C at the beginning of the PCR.
• Potassium xanthogenate method, Peter Young in York
• Forensic kits, e.g., punch cards

CTAB DNA Extraction