Team:METU Turkey/Results Discussion/PCR Purification

From 2010.igem.org

Latest revision as of 03:49, 28 October 2010

MATERIALS

- Heating block
- Microcentrifuge

- PCR Purification Kit

  
- Binding Buffer
  
- Wash Buffer
  
- Elution Buffer 

- Water nuclease free
- Purification columns
- 1.5 or 2 mL microcentrifuge tubes
- Ethanol 96-100 %.
- 3 M sodium acetate, pH 5.2

SOLUTIONS

- Prior to the initial use of the PCR Purification Kit, dilute the Wash Buffer (concentrated) with ethanol (96-100%)
- Examine the Binding Buffer for precipitates before each use. Dissolve the precipitate by warming the solution to 37 C and cooling to 25 C.
- Wear gloves when handling the Binding Buffer
- Check the color of the Binding Buffer solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 uL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.

CHECK-LIST PROCEDURE

- Add a 1:1 volume of Binding Buffer to completed PCR mixture. Mix by pipetting gently up and down.
- If the DNA fragment is <500 bp, add a 1:2 volume of 100 % isopropanol [optional]. Mix by pipetting gently up and down.
- Transfer up to 800 uL of the solution from epp to the purification column.
- Centrifuge for 30-60 sec at >12000 g.
- Discard the supernatant from collection tube. [If the total volume exceeds 800 uL, the solution can be added to the column in stages. After the addition of 800 uL of solution, centrifuge the column for 30-60 sec and discard flow through. Repeat until the entire solution
has been added to the column membrane.]
- Add 700 uL of Wash Buffer (diluted) to the purification column.
- Centrifuge for 30-60 sec at >12000 g.
- Discard the flow through and place the purification column back into the collection tube.
- Centrifuge the empty purification column for an additional 1 min at >12000 g to completely remove any residual wash buffer.
- Transfer the purification column to a clean 1.5 mL microcentrifuge tube.
- Add 50 uL of Elution Buffer to the center of the purification column membrane and centrifuge for 1 min at 12000 g.
- Discard the purification column and store the purifi ed DNA at -20 C.

TROUBLESHOOTING

- For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 uL does not significantly reduce the DNA yield. However, elution volumes less than 10 uL are not recommended.
- If DNA fragment is >10 kb, prewarm Elution Buffer to 65 C before applying to column.
- If the elution volume is 10 uL and DNA amount is >5 ug, incubate column for 1 min at room temperature before centrifugation.