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Plasmid calculation question (Standard Curve Quantification)

Standard Curve Quantific qPCR TOPO plasmid

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#1 Luiz Fernando Lino

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Posted 29 July 2014 - 06:42 AM

I need help to building my standard curve. I will use plasmid TOPO, but I don’t know what kind (pCR 2.1 TOPO or pCR II TOPO). The question is: They have different length (3.931nt and 3.973 nt respectively) and showed differents results on the number copies calculation ( 7,31x10and 7,26 X10 respectively). Will I have problems in my absolute quantification? What plasmid should I take?



#2 bob1

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Posted 29 July 2014 - 12:52 PM

The best thing you could do is work out which plasmid you have - this should be possible by sequencing portions of the plasmid and comparing to known sequence.  Incidentally, the plasmid isn't called TOPO - it is called pCR2 or pCR2.1.  TOPO indicates that it has topoisomerase added to a linearized form that can be used for TA cloning, hence there are many different plasmids called pXXXX-TOPO. Once you have cloned something into the plasmid using the TOPO system, TOPO is no longer used in the name.



#3 Trof

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Posted 29 July 2014 - 01:17 PM

Those plasmids have almost identical lengths, as you can see, from the copy number calculation as well.

But you are not using empty plasmids as standards are you, these are empty vector lengths? 

 

You are going to use plasmid with the length of vector+your insert. Don't forget to use the complete legth for the calculation of copy number.

 

Next thing.. "showed differents results on the number copies calculation". Copy number calculation from what?? You need concentration and basepair length to measure a copy number. Where you get a concentration calculation? The empty TOPO vector has linked enzyme bound to it, any DNA measurements prior transformation will be screwed.

 

Actually it doesn't matter at all, what kind of plasmid you use for the construction of a standard, set aside that you are comparing two almost identical ones. You can make standard of any plasmid, of any sequence that has a defined legth, is stable and you know its concentration.

 

Better look up proper was to treat a plasmid to make it a precise standard (single cut digest and purify and mix with dummy nucleic acid to create similar complexity as genomic DNA - if your template would be genomic DNA) than wonder about plasmid lengths.

 

As for the question if you will have problems in your quantification, no one can tell you that.


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.

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#4 Luiz Fernando Lino

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Posted 29 July 2014 - 02:58 PM

"But you are not using empty plasmids as standards are you, these are empty vector lengths? "

 

My insert was the complete genome of porcine circovirus 2 (1768nt)

 

After the extraction of  plasmid, I used quantification with quBit 2.0 ( using high sensitivity). Result: 44,9 ng/uL

 

I used this equation:

 

 

44,9*10-9g/uL DNA * 6,022 *1023

vector + insert(1768) * 649

 

Results

 5699 nt  or 5741nt[ insert + vector]

 

 

"Actually it doesn't matter at all, what kind of plasmid you use for the construction of a standard, set aside that you are comparing two almost identical ones. You can make standard of any plasmid, of any sequence that has a defined legth, is stable and you know its concentration."

 

I found differents copies number of standard (insert)7,31x10or 7,26 X109. My question is if this differences will super ou sub estimate my comparasions with test samples in absolute quantification.

 

thanks,



#5 Trof

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Posted 29 July 2014 - 11:55 PM

The calculation is right, you need to use the complete length.

You can use online tools to calculate next time, this one or any other.

 

So you have your insert in two different plasmids? Or you don't know what kind of plasmid do you have?

 

In any case, it doesn't matter which one you use if you have two.

If you calculate with a different plasmid length than you really have you can create a small mistake, but if you have no way how to identify what type of TOPO vector was used, there is nothing you can do about anyway. The differences are very small, it is likely that further dilution will bring more errors to it, so that this wouldn't matter.

 

You will need to do a serial dilution of your plasmid standard (like 109, 108, 107, 106 105, ...), to have a complete range inbetween witch your unknown virus sample would fall (you need to find this in the literature or by experience). 


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


#6 Luiz Fernando Lino

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Posted 30 July 2014 - 08:05 AM

Trof: You are correct : I don't know what kind of plasmid I have.

 

I have 3 options:

 

Sequencing ,

enzyme restriction assay

or just ignore this possible error.

 

What do you think I should to do, considering my goal.(standard curve quantification).



#7 Trof

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Posted 31 July 2014 - 10:27 AM

Ignore it. Or use a "mean" size, so you will know that in any case you have a incredibly small error, but 1/2 smaller than if used calculation for a different vector.

 

The absolute quantification is not that precise anyway that it would matter, you will quite sure bring other errors into it, in dilutions, etc. Nothing is 100% accurate.


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






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