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pcr purification doubt


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4 replies to this topic

#1 siddharthsameer

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Posted 16 August 2012 - 04:45 AM

hello all
, i need ur suggestion,, i am doing my master thesis , I had my perfect pcr but at the time of pcr purification my supervisor is asking me to use only the volume that can hold only 10µg, as it is mentioned in the pcr purfication kit of qiagen, but i really do not understand why cant I purify the whole product...can u give me a sloid reason so that i can convience my supervisor to do what i want..
And the last time also they asked me to do the purification in the same manner and did not work...and again they are asking me to do the same, but I dont want to do it.. kindly reply me soon..
thanks and regards

#2 hobglobin

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Posted 20 August 2012 - 08:48 AM

The supervisor surely asked you to do this, because the DNA binding capacity of the Qiagen columns is limited to 10 µg. If you use more DNA it would just flow through and is lost in the eluted fluid (if you don't collect it and clean it with another column). This means if you need more DNA you should split your sample and use as much columns as needed for your DNA amount...question is if you need more DNA than the 10 µg...
For the second question more information is needed...
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#3 ascacioc

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Posted 20 August 2012 - 09:50 AM

more than 10ug out of a PCR reaction? the supervisor probably measures the concentration of the DNA after the PCR reaction (without PCR clean-up) which includes also the unincorporated dNTPs; how else would he 'know' that there are more than 10 ug DNA? Solution: just do the clean-up with what you get out of the PCR. You will see that you don't get more than 1.5 ug (best case scenario).

Andreea

#4 phage434

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Posted 20 August 2012 - 02:25 PM

What she said.

#5 hobglobin

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Posted 21 August 2012 - 08:07 AM

yup...didn't thought about that....
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.




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