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Question about Plasmid Isolation Protocol containing Phenol/Chloroform PEG


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#1 hans

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Posted 18 December 2009 - 02:35 PM

Because of Problems with Column Isolation (not cleared enough) i want to try the old method of Plasmid DNA Purification by PEG Preciptation.

After the 3 Steps of Lysis and neutralization with the 3 common buffers (EDTA/TRIS/Gluc, 0.2 NaOH SDS1% +RNase, 3M Potassiumacetate) and centrifugation
the next step of the protocol is: Plasmid DNA Extraction with buffered Phenol, then with 24:1 chloroform/isoamyl alcohol.

-->How much of the Phenol do is use (same volume as supernatant after centrifugation ?)
-->I guess there will be then 2 Phases? Do i centrifuge before adding 24:1 Chloroform/isoamyl and after that? Which Phase is the right one (the one above?)

After this step i am supposed to ad 1/4 Ammonium acetat to aqueous phase and mix
-->??? what phase???

than add 2 vol 100% ethanol und place in dry ice
Then Centrifugation
Than wash with 70% ethanol


PEG Preciptiation
resuspend the pellet in 2 ml Te buffer and add 0.8 ml PEG
Incubation for 15hr at 0C
Recover DNA bei Centrifugation
Resuspend Pellet in TE buffer
Ethanol precipitation by 3M Sodium acetat

--> Last step? how much of the ethanol and how much of the sodum acetat??

Thanks for any help!!!

hans

#2 bob1

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Posted 21 December 2009 - 03:46 PM

the phenol step is to dissolve the left over protein in the extract, and pH buffer to extract DNA as opposed to RNA. You should add phenol, mix, then add the chlorofom/isoamyl, mix again and then spin to separate the phases. You want to retain the aqueous phase, which will be the upper phase, unless you are using tiny volumes (<50 ul) of extract compared to P:C:I.

I have attached my protocol, which is taken from the ABI sequencing manual. It is quite clear with all volumes and steps, and does away with phenol. Steps 1-7 are standard alkaline lysis proceedure, use whatever solutions you normally use.

Attached Files



#3 hans

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Posted 28 December 2009 - 02:27 AM

Thanks for your help! it worked quiet well!!
Do you know if the final plasmid is clean enough for sequencing and transformation?
if not what should i do to obtain clean quality?

#4 hanming86

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Posted 30 December 2009 - 08:33 PM

Thanks for your help! it worked quiet well!!
Do you know if the final plasmid is clean enough for sequencing and transformation?
if not what should i do to obtain clean quality?



For transformation, definitely . sequencing i would assume it's alright. washing with 70% ethanol usually will provide sufficiently clean DNA. way better than conventional column purification.
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#5 bob1

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Posted 04 January 2010 - 05:36 PM

Yes, definitely good enough for sequencing and transformation.




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