Please record all extractions correctly in the database. See database protocols for isolations for more information.

Sigma Extract-N-Amp

Spin-down protocol (for beetles)

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

With BSA as a binding agent

• beetle body parts (legs work well from specimens killed with 95% EtOH)
• 40uL of Extraction Solution per sample (in yellow tray in the freezer)
• run protocol Ex-n-Amp on thermocycler (96C for 30 minutes)
• add equal volume(to extract solution) of 3% BSA (will bind extra stuff)— (40uL of Extraction Solution:40uL of BSA)
  • vortex thoroughly
• spin down, store the upper half (30uL) as your final sample
• use the 0.5-1.0uL of this supernatant for PCR

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

 

General Ex-N-Amp protocol for fungi:

1. Prepare strip tubes with 40uL of Sigma Aldrich extraction solution. This can be found in aliquots in the shared box in the door of the small freezer.
2. Scrape approximately 10uL of hyphae from fungal colony using a sterile pipette tip (20uL works best and is not used for much else) or scalpel.
3. Add fungal material to extraction solution and vortex.
4. Run Ex-N-A protocol on a thermocycler (96C for 30 minutes).
5. Spin down with high rpm.
6. Pipet off the top 25uL: that’s your extract. It is often safe to dilute this a bit if you will need more than 25 uL, but this is not standard procedure.

Note: If BSA is needed, we have a stock of 2% molecular grade BSA in our freezer. This should be made into aliquots before using for samples. This is not standard, and only used in cases where it increases DNA amplification success.

OLD: Spin-down protocol (for fungi) 

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

With BSA as a binding agent

• 10uL of original sample
• 20uL of Extraction Solution per sample (in yellow tray in the freezer)
• run protocol Ex-n-Amp on thermocycler (96C for 10 minutes)
• add equal volume(to extract solution) of 3% BSA (will bind extra stuff)— (20uL of Extraction Solution:20uL of BSA)
  • shake thoroughly
• spin it down, store 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%

 

Manufacturer’s protocol – DON’T USE, the Dilution Solution inhibits PCR

With genuine Sigma components. Sigma manual here.

1. 20uL of Extraction Solution per sample
2. 96C for 10 minutes
3. add equal volume of Dilution Solution
4. ready for PCR. Allegedly stable for several months (says Sigma).

 

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

 

CTAB DNA Extraction