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white residue after DNA purification


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#1 supernater

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Posted 17 December 2010 - 08:12 AM

I am purifying a plasmid using alkaline lysis. I precipitate the DNA using 0.7x the volume of isopropanol and I wash with 70% ethanol and air dry for 10 minutes. When I resuspend the pellet in water I keep getting some white precipitate that won't dissolve. I know that my plasmid DNA is suspended because I can measure it on the spectrophotometer and see it on the gel. Is this white precipitate protein? Am I using too much isopropanol?

#2 cdugard

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Posted 17 December 2010 - 08:23 AM

Let it dry longer. I've found if you can see any moisture in the tube at all before you try to resuspend it, the precipitate is incredibly hard to dissolve. Letting it dry until the pellet is clear will help you dissolve it almost completely.

#3 Michaelro

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Posted 17 December 2010 - 08:33 AM

Hi
I don't know the exact identity of the precipitate but in my case, it never caused any troubles in subsequent reactions given that ethanol was completely removed.
Remember that it is not column purification so you cannot expect 100% purity.
Good luck
Michael

#4 bob1

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Posted 18 December 2010 - 07:28 PM

White stuff is protein most likely, it shouldn't affect PCR (though it may if there is enough of it) and your plasmids are not pure enough for transfection into mammalian cells or for sequencing.

You can clean up this precipitate by pelleting with 13% PEG-8000 as below - from step 13 if you have chloroform extracted already.

To prepare plasmid DNA by alkaline lysis/PEG treatment:
Step Action
1 Pellet 1.5-mL aliquots of culture for 1 minute in a microcentrifuge at maximum
speed.
Note A total culture volume of 4.5 mL can be spun down per tube without
changing volumes in the procedure. This allows you to achieve a threefold increase
in yield while eliminating the need for extra tubes and additional handling.
2 Remove the supernatant by aspiration.
3 Resuspend the bacterial pellet in 200 μL of GET buffer by pipetting up and down.
4 Add 300 μL of freshly prepared 0.2 N NaOH/1% SDS. Mix the contents of the tube
by inversion. Incubate on ice for 5 minutes.
5 Neutralize the solution by adding 300 μL of 3.0 M potassium acetate, pH 4.8. Mix by
inverting the tube. Incubate on ice for 5 minutes.
6 Remove cellular debris by spinning in a microcentrifuge at maximum speed for 10
minutes at room temperature. Transfer the supernatant to a clean tube.
7 Add RNase A (DNase-free) to a final concentration of 20 μg/mL. Incubate the tube
at 37 °C for 20 minutes.
8 Extract the supernatant twice with chloroform:
a. Add 400 μL of chloroform.
b. Mix the layers by inversion for 30 seconds.
c. Centrifuge the tube for 1 minute to separate the phases.
d. Transfer the upper aqueous phase to a clean tube.
9 Add an equal volume of 100% isopropanol. Mix the contents of the tube by
inversion.
10 Spin the tube in a microcentrifuge at maximum speed for 10 minutes at room
temperature.
11 Remove the isopropanol completely by aspiration.
12 Wash the DNA pellet with 500 μL of 70% ethanol. Dry under vacuum for 3 minutes.
13 Dissolve the pellet in 32 μL of deionized water.
14 Add 8.0 μL of 4 M NaCl, then 40 μL of autoclaved 13% PEG 8000.
15 Mix thoroughly, then leave the sample on ice for 20 minutes.
16 Pellet the plasmid DNA by spinning in a microcentrifuge at maximum speed for
15 minutes at 2–6 °C.
17 Carefully remove the supernatant. Rinse the pellet with 500 μL of 70% ethanol.
18 Resuspend the pellet in 20 μL of deionized water. Store at –15 to –25 °C.

Edited by bob1, 18 December 2010 - 07:32 PM.





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