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PCR with Plasmid recovered from filter paper


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6 replies to this topic

#1 Indhumathi

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Posted 26 March 2013 - 08:59 PM

Hi,
I have got a plasmid on filter paper. After recovery, can I use it directly for PCR? I read in a blog that it should only be used for transformation.
Indhu

#2 leelee

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Posted 26 March 2013 - 11:17 PM

I suspect that the main reason the blog suggested using it only for transformation is that you will then have a glycerol stock of bacteria containing the plasmid that you can go back to time and again to produce more plasmid if and when you need it.

Rather than using it all up for PCR and then having to send for more when you run out. Or contaminating your single aliquot while using it for multiple PCRs etc.

I can't see any reason why you wouldn't be able to use it for PCR. I routinely run QPCR on DNA samples in saliva collected onto filter paper. I actually just put a little punched circle of the paper directly into my master mix and run. There are specific tools available for this purpose, if you need to be doing a high number of these.

If I were you, I would elute into water then use a few ul to transform into bacteria and a few ul for your PCR, then store the rest as a back up until you have verified your transformation was successful and you have isolated and verified the plasmid from your new stock.

#3 Indhumathi

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Posted 27 March 2013 - 09:02 PM

hi leelee,
Thanks for the reply. I am planning to use part of it for transformation and part of it for PCR to confirm the presence of the required portion in the plasmid before going for transformation. The sender did not mention the amount of plasmid present in the circle. I read somewhere it usually would be around 2 ug. How much of sterile water I should use to extract the plasmid?

#4 badguy

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Posted 27 March 2013 - 11:25 PM

you can use 50ul of TE buffer/nuclease free water to elute the plasmid.
It's better to do transformation rather than doing PCR directly. You never how much of plasmid has been loaded into the paper.
Is the plasmid in FTA card? There are instructions on their website on how to elute plasmid from it.

#5 Indhumathi

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Posted 31 March 2013 - 09:27 PM

hi
Thanks for your reply. plasmid is not in FTA card, its just in filter paper.

#6 badguy

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Posted 01 April 2013 - 04:48 PM

If that is the case, just elute the plasmid with 50ul of TE buffer or less by suspending and centrifuging at full speed if my memory serves me right.
after that just add in your competent cells and do the transformation.

#7 Indhumathi

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Posted 01 April 2013 - 10:39 PM

thanks again for the reply.




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