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Plasmid Linearization Problems

linearization plasmid TOPO Hind III standard quantification

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9 replies to this topic

#1 Luiz Fernando Lino

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Posted 05 August 2014 - 02:00 PM

I realized a plasmid extraction with miniprep kit (FERMENTAS).

Yield: 44,9ng/uL by qubit 2.0

 

 

 

So I realized a restriction assay with Hind III for linearization of my standard. I followed the manufacture's recomendations of the enzyme.

I used 10uL of plasmid sample [10 *44,9], with final volume of 20uL

 

 

 

So for gel purification i tried 8% agarose gel + 7uL of my sample (linearized - " I expected").

Run time: 1:20h

Expected size = 5700 pb (vetor + insert)

 

 

But I can't find nothing in my gel.

 

Questions: Is this concentration [44,9 ng/uL] a problem? Have I to try another extraction for a better yield?

 

 

 



#2 bob1

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Posted 05 August 2014 - 02:56 PM

You should be able to see 450 ng of plasmid on a gel without any difficulty.

 

45 ng/ul is quite low for these sorts of preps - try running some uncut on a gel and see if you can see anything.



#3 phage434

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Posted 05 August 2014 - 07:37 PM

Did you really mean an 8% agarose gel? I'm not sure you can actually make one. I hope that was 0.8%

You only ran about 150 ng on the gel, according to your measurements, but even that should give a blindingly bright band.

Did your ladder run correctly?



#4 Luiz Fernando Lino

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Posted 06 August 2014 - 03:44 AM

Veteran

 

Ok, sorry did i mean 0,8% agarose gel.

 

But I used 7uL on gel. [7*44,9ng] = total of 314ng

 

My ladder run at 1:20h, what about this time for the plasmid size: 5700pb?



#5 phage434

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Posted 06 August 2014 - 04:37 AM

No, you have diluted your DNA in the restriction reaction, by a factor of two (10 ul into 20 ul reaction) and then used 7 ul of that reaction, so you have only half as much as you thought (3.5*45 ng).

But that is not your problem. Was your ladder visible on the gel when you imaged it?



#6 Trof

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Posted 06 August 2014 - 04:47 AM

It's hard to say from the time only, does your ladder have bands covering the desired range (around 4 - 6 kb)? If so, you should know where to look for your band.

 

In any case, cut or uncut, 300 ng of ~6 kb plasmid would be always visible on gel, unless you forgot to stain it.

Did you observe any bands elsewhere or a bright smear or a bright patches remaining in the gel? The DNA has to be somewhere.

 

Are you sure your vector is cut only once by HindIII? If for example it cut many times and there would be many little fragments, their divided concentration would dilute so much, that they may not be visible on the gel. That is the only circumstance I can imagine, when 300 ng would not be seen on gel anywhere.

 

Regarding the restriction, your DNA comprises half of the reaction volume. That may or may not be a problem with some enzymes, as DNA should be only around 1/4 of overal volume, because it can contain additional salts affectiong enzyme activity (or star activity). Not sure if that's a case HindIII too.


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#7 Luiz Fernando Lino

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Posted 12 August 2014 - 05:08 AM

In the second time I realized:
 
3,5 water
2,0uL buffer
14uL plasmid DNA [44,0ng/uL]
 
Dnase inactivation : 80C at 10min., folllowing by add of 0,5uL HindIII
 
total volume 20uL
 
On the 1% gel i used: 20uL of "linear" plasmid and 2,5uL of supercoiled plasmid as control.
 
Results:

 

https://scontent-a-l...638209901_n.jpg

 

Linear plasmid degraded and supercoiled plasmid

 

Any sugestions?

thanks



#8 phage434

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Posted 12 August 2014 - 05:21 AM

Your "control" DNA lane shows little plasmid DNA. I suspect your estimate of the amount of DNA  you are working with is far off.

Several potential issues:

* You should never do a digestion in which the majority of the volume is DNA. Here, 14 ul of the 20 ul is "DNA" which likely has many other things, such as Gu-HCl from preparation, or ethanol. These other chemicals can inhibiit or change the restriction digest by enzymes. I'd suggest no more than 25% of your final volume be DNA.

* Your bands become dimmer as they become shorter, because there is less DNA in them. So, if you start out with a single quite faint band (as here) then cut it, the bands may become invisible. I think this is what is happening here.

* Your DNA amounts are quite small. Likely the preparation of the DNA is at fault. It appears to be a large plasmid. If it is low copy, then you likely need to prepare something more than a miniprep, and concentrate the DNA after the prep.

You can be fooled by instruments that claim to measure DNA, such as a nanodrop. Trust the gel more than those.



#9 Luiz Fernando Lino

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Posted 12 August 2014 - 08:16 AM

I used 14uL because my plasmid concentration is 44,9 ng/ul and 23ng/ul, very poor.

 

how much uLs should i use in these cases?



#10 Trof

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Posted 12 August 2014 - 09:18 AM

And ybout that "DNase inactiovation".. I'm not sure why you do that, if there were DNases in the DNA, you wouldn't help a bit, if there are in water of buffer.. well they shouldn't be there at all in the first place...

 

But I faintly remeber something, that salts and higher (> 60 deg) temperature degrades nucleic acids.. I know that RE inactivation steps are quite commonly used after restriction, but I see no point in doing it before restriction anyway..

 

As for plasmid concentration. If it's such low, you need to concentrate it, usually precipitation and dissolving in smaller volume afterward.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






Also tagged with one or more of these keywords: linearization, plasmid TOPO, Hind III, standard quantification

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