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Maxiprep Question - (Sep/21/2005 )

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Hi all,


I just did maxiprep today and after I calculate the DNA concentration using spectrometer I got 1057 microgram/mL and it also said that the protein concentration is -812microgram/mL...

I'm just curious about why I got negative result in protein concentration....is anyone know what happened with my maxiprep DNA? Is my result is ok?

Thank you in advance.... smile.gif

-verencia-

check 1┬Ál on agarose gel to see what your extract looks like...
check if the UV lamp in in function. If it's broken, the readed OD will be very high, leading to such concentrations.
And finally check the measurement, by a know solution prepared one month earlier.
after doing the baseline, do you make a measure to check if the OD is at the baseline?
fred

-fred_33-

What is your 260/280 ratio? This is more meaningful.

-tap14-

Hi all, thx for the reply...

I use 260/280 ratio for this spectrometer...

I asked my boss and she said that 1057 is the normal DNA concentration for maxiprep but I'm still curious about why the protein concentration is -800 microgram/mL....

In my maxiprep end result I saw 2 layer, anyone knows if this normal??

I'm new in this field so I'm little confused about this.... unsure.gif

Thanks

-verencia-

"I use 260/280 ratio for this spectrometer"


blink.gif
So what is it?

-tap14-

QUOTE (tap14 @ Sep 22 2005, 12:29 PM)
"I use 260/280 ratio for this spectrometer"


blink.gif
So what is it?


Do you mean the dilution ratio??
it's 1:50

-verencia-

Divide the OD reading at 260nm and OD at 280nm. If your DNA is pure, you would expect 1.8-2.0. If <1.8, then you most likely have protein contamination in your prep.
How did you determine your protein concentration?

-tap14-

QUOTE (tap14 @ Sep 22 2005, 01:13 PM)
Divide the OD reading at 260nm and OD at 280nm. If your DNA is pure, you would expect 1.8-2.0. If <1.8, then you most likely have protein contamination in your prep.
How did you determine your protein concentration?


Actually in spectrometer machine which I used it automatically calculate the protein concentration in addition of DNA concentration.....

Tap14, do you mean that it is weird to have -800ug/mL protein concentration?

thx

-verencia-

The 260/280 DNA ratio is 1.760

but after I elute the DNA with EB buffer and spin it I got 2 layer solution (more viscous solution on the bottom)......

-verencia-

QUOTE (verencia @ Sep 23 2005, 02:50 AM)
but after I elute the DNA with EB buffer and spin it I got 2 layer solution (more viscous solution on the bottom)......


This is a problem. A two-layer solution just from a MaxiPrep, as eluted? You didn't vary the protocol at all?

I would check a bit from each layer to find where the DNA is. Discard the other layer, and clean up the remainder with either a PCR clean-up kit or a gel extraction kit.

-HomeBrew-

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