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Guanidine isothiocyanate in PCR


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#1 Trof

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Posted 30 January 2014 - 02:18 AM

I need to do PCR on human cell genomic DNA, very small amount (around 5000 cells) that were frozen in 500ul QIAGEN RLT buffer (not that I could do anything about that).

 

Now RLT is proprietary, but supposedly contains a high concentration of guanidine isothiocyanate and some other nasty things. 

 

My original idea was just lyse the cells by heating and just put into PCR, howevrer I got most of them in Trizol (isolation required) or RLT. I can isolate from Trizol thought that is a pain in the ass with DNA and the small number of cells is problematic (I've done that before, but repeatedly thought of better way to do that), but I can't even use any standard isolation protocol from RLT and some Q colums I have, because it's generaly designed for RNA isolation. In addition, elution volumes from columns are to high. I'm kind of affraid I wouldn't get any DNA, from whole volume used (which I idealy should not use whole).

 

So I'm thinking of some quick way how to get PCR-suffucient DNA out of the RLT solution, or at least minimize the guanidine content. Pelet the lysate on high g (if possible) and take the GITC out? Compensate the PCR reaction for GITC and use only a very small amount?

 

Any other ideas?


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


#2 Trof

Trof

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Posted 31 January 2014 - 09:16 AM

Just for the record.

 

I took 200 ul of the RLT, precipitated with the same volume of isopropanol for 10 minutes, centrifuged 15 min/16 000g/4 deg (probably could be less), washed pelet (completely invisible) with 200 ul of 100% EtOH and 70% EtOH (centrifuged inbetween as aforementioned), dissolved in 10 ul of TE, used 1 ul of this to PCR (with BSA [10 ug] only or BSA+0,8M betaine) and got pretty nice bands (betaine slightly better), definitelly enough for my purpose :)

 

Using 1 ul of RLT as is in the reaction, gave no product.


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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