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pcr purification trouble

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pcr purification trouble

Postby siddharthsameer » Sat Aug 11, 2012 8:10 am

Hello all
I am into a trouble need some suggestion to correct myself.
I had my pcr and I used 2 step pcr and i observed the band on agarose my desired sequence. Measured the concentration before and after pcr purification, but the concentration got down considerbaly. I did not purify the whole amount of PCR product , just the 10microgram according to the binding capacity of the purification kit column. and then I proceeded wit the further step but did not get any result of the transformation,
I am starting again from the pcr , I would like to know shall I purify the complete volume of PCR product or the just the voulme of 10microgram as I did last time.. The volume of my PCR was 50 microlitre.. kindly help me....
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Postby JackBean » Sun Aug 12, 2012 12:33 pm

I don't see reason why not purify everything. The worst case? You overload the column, but it's still better than getting nothing, right?
How did you measure the amount of DNA? By UV?
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Re: pcr purification trouble

Postby siddharthsameer » Sun Aug 12, 2012 3:57 pm

hi
I measured the concentration through UV...so i am doing again PCR and this time I have decided to load all the sample for purification...
Another thing I would like to ask,is that i already of the used up my vector, and I plated the vector on one of the lb plate which i used as a negative control at the time of doing ligation,,So i would be trying to make the preculture from that and then the main culture and would do the miniprep to get my vector..I can easily know the concentration through UV but how do I get the mass of my vector...My vector is pPICZalphaA ....
since i am doing all this thing for the first time, so kindly help me.....
regards
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Postby JackBean » Sun Aug 12, 2012 8:38 pm

the thing is, that in UV you measure everything that absorbs at 260 nm, including proteins, but mainly the nucleotides, which are in pretty high concentration in PCR reaction, right? So instead of purification of your product, you were purifying nucleotides ;)
If you want to check your PCR, you have to use either fluorescence or agarose electrophoresis.

Sorry, I didn't get the other thing. But if you want to calculate mass of your vector, you need to know how big is it. You should easily find map of the vector with size in kbp, just add your insert and that's it.
http://www.biolib.cz/en/main/

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Re: pcr purification trouble

Postby triton » Wed Aug 22, 2012 7:27 pm

siddharthsameer wrote:Hello all
I am into a trouble need some suggestion to correct myself.
I had my pcr and I used 2 step pcr and i observed the band on agarose my desired sequence. Measured the concentration before and after pcr purification, but the concentration got down considerbaly. I did not purify the whole amount of PCR product , just the 10microgram according to the binding capacity of the purification kit column. and then I proceeded wit the further step but did not get any result of the transformation,
I am starting again from the pcr , I would like to know shall I purify the complete volume of PCR product or the just the voulme of 10microgram as I did last time.. The volume of my PCR was 50 microlitre.. kindly help me....


Hi,
I do not use the kit at all. Just run the gel with say 10 microliters and check if there is a single band. Then you have two ways you can walk. EITHER use pcr product directly for ligation reaction OR add 0,3ul of ExoI and 0,3ul of CIP, incubate it in a cycler for 30 minutes at 37 degrees of celsius and then 20 minutes at 80 degrees of celsius. Use the purified PCR product directly for a sequencing reaction. If your vector is expression vector, use the way number 1, if you want just a sequence, forget about the vector and use the way number 2.
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