Freimoser:Protocols:PolyP1: Difference between revisions
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Revision as of 16:25, 5 October 2006
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General comment:
Poly P can be purified on Qiagen PCR-product purification columns (either individual spin columns or for example 96-well plates). The columns can be reused after recycling according to the protocol given below.
Extraction and purification of poly P from yeast
- Pellet 0.5 or 1 OD600 Unit of yeast cells in a 1.5 ml Eppi. (12000 rpm, 1 min), discard the supernatant.
- Add exactly 50 μl of 1 M sulfuric acid and vortex vigorously for 15 seconds (This extract can be stored at -20°C if necessary).
- Add exactly 50 μl of 2 M NaOH to the extract to neutralize the pH.
- Add 100 μl of 1M Tris-Malate buffer pH 7.5 with 6% Neutral red solution as an indicator for the pH. The solution should be red or dark orange. (neither pink nor yellow)
- Centrifuge (13000 rpm, 1 min) and transfer the supernatant to a new tube.
- Add 600 ul of 6 M NaI and mix.
- Fill the mixture in the purification plate and apply vacuum for 1 min.
- Wash with 750 ul of washing buffer and apply vacuum for 1 min.
- Repeat this step, but apply vacuum for 10 min.
- Remove residual washing buffer on outside of plate.
- Add 100 ul of ddH2O (which must not contain any phosphate!) and let the plate stand for one minute before elution by vacuum for 2 min.
- After use wash columns with 1 ml ddH2O.
Quantification of poly P
- Prepare a master mix including 10 ul of 10X buffer, 1 ul (0.2 ug) of Polyphosphatase from S.cerevisiae and 39 ul of phosphate free ddH2O for each sample.
- Fill 50 ul of this master mix to a 96 well flat bottom microtiter plate and add 50 ul from your eluted iPoP samples.
- Incubate for 1-2 hours at 37°C.
- Prepare a standard curve for Pi detection: Add 0, 5, 10, 20, 40, 60, 80 and 100 ul of 100 uM NaH2PO4 (Pi) to your microtiter plate and fill up to 100 ul with ddH2O.
- Prepare a 1:3 dilution of your samples: Add 50 ul of ddH2O to your samples, mix by pipetting and transfer 50 ul to a new microtiter plate containing 100 ul ddH2O per well. Mix again by pipetting and discard 50 ul solution from the diluted samples. Do the same to your Pi standart curve.
- Mix 86 ul of Molybdate solution and 64 ul of Malachite green solution per sample and add 150 ul to each sample.
- Use a 96 plate reader to determine the OD at 595 nm. (590 – 650 is a possible range)
Recycling of Qiagen-columns for poly P purification
- Add 500 ul of 0.2 M Acetic acid (0.6ml in 49.4ml ddH2O) and apply vacuum for 1 min.
- Add 500 ul of 0.05 M EDTA pH 8 and apply vacuum for 1 min.
- Add 1 ml of ddH2O and apply vacuum for 1 min.
- Repeat last step.
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