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plasmid purification

nanodrop vs. agarose gel

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4 replies to this topic

#1 nightingale

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Posted 22 July 2019 - 07:17 AM

Dear All,?
?
kindly follow below :-
?
1) Low copy number plasmid.
2) qiagen maxi kit purification.
?
a first trial results ...
?
?
gel below would show :-
?
sample (1) :
well 1: clear lysate containing supercoiled and open circular plasmid DNA and degraded RNA.
well 2: degraded RNA only, depleted from plasmid DNA that is bound to the coulum.
well 3: wash-through containing traces of RNA no plasmid DNA
well 4: pure plasmid DNA?
?
sample (2) :
well 5: clear lysate containing supercoiled and open circular plasmid DNA and degraded RNA
well 6: degraded RNA only, depleted from plasmid DNA that is bound to the coulum.
well 7: wash-through containing traces of RNA.
well 8: pure plasmid DNA
?
sample (3) :
well 9: clear lysate containing supercoiled and open circular plasmid DNA and degraded RNA
well 10: degraded RNA only, depleted from plasmid DNA that is bound to the coulum.
well 11: wash-through containing traces of RNA.
well 12: pure plasmid DNA
?
sample (4) :
well 13: clear lysate containing supercoiled and open circular plasmid DNA and degraded RNA
well 14: degraded RNA only, depleted from plasmid DNA that is bound to the coulum.
well 15: wash-through containing traces of RNA.
well 16: pure plasmid DNA
?
you can see that samples 1 and 3 were successful.
?
i read? samples in : well (4), well (8), well (12), well (16).
on nanodrop.
and the results in all were around 100ng/ul, even wells 8 and 16.
?
is this normal ??
?
?
4 segments.jpg

Edited by nightingale, 22 July 2019 - 08:12 AM.

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#2 OldCloner

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Posted 22 July 2019 - 03:36 PM

Hi Nightingale,

 

It does seem that only your 1st and 3rd preps worked, but it looks like there was starting material in all four.  For numbers 2 and 4, were you able to see pellets in your centrifuge tubes when you did the final precipitation and 70-percent ethanol wash? You have to be careful not to lose a pellet during the wash step- they are clear and very hard to see after the 70-percent wash, and usually they have moved from where they originally stuck. Are you sure you got all the pellet resuspended? Sometimes they don’t go into solution well and remain stuck on the side of the tube after you dry them.  If you “overdry” them, they are hard to get dissolved into your TE (or whatever you use to re-suspend them.)  How large are these plasmids (in kb)? Larger ones are harder to dissolve. I used to do this step at 50 degrees C to help the DNA go into solution.

 

I made a habit of not discarding (or washing) my last centrifuge tubes until after I had evaluated my DNA on a gel, in case I left DNA behind stuck to the wall. I could always go back and try to re-dissolve any leftovers from the tube wall with fresh buffer if necessary.  What kind of centrifuge tube are you using ( ex. Oak Ridge type? Made of?)

 

I have no idea why all your Nanodrop readings seem equal. No, not normal.

 

Hope my questions help you trouble-shoot.


Edited by OldCloner, 22 July 2019 - 03:38 PM.


#3 nightingale

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Posted 23 July 2019 - 04:06 AM

thank you OldCloner smile.png

 

the preps vary in sizes ; my empty plasmid is 3kb.

and each of the 4 preparations has at least a 1kb plus size.

 

yes its obvious that preps 2 and 4 failed, and i didnt see a pellet in both ...

but i can repeat them, not an issue to worry about.

regretfully i discarded the last-step tubes ,,, i should have stored them ... knowing that the first step and the following 2 steps were all successful.

but i will next times, thank you.

 

 

the tubes are oak ridge type yes, made of PPCO.

 

my concern now is that why are the nanodrop readings like that!

i learned its not calibrated. 

so am waiting for the calibration kit to be delivered.

 

your tips always help! thanks a bunch.


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#4 OldCloner

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Posted 23 July 2019 - 10:39 AM

You're welcome-I think you are on the right track.  Our tubes were Oak Ridge- FEP: probably very similar to PPCO in that, although translucent, neither material is crystal clear, so pellets are hard to see.

 

Hope your new preps work out!



#5 nightingale

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Posted 25 July 2019 - 05:29 AM

thank you! 


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