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protocol to relieve melanin inhibition of PCR


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#1 crom80

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Posted 23 October 2013 - 10:55 AM

Hi,

 

I performed PCR amplification of genomic DNA isolated from human primary melanocytes.

I got no bands, positive control worked fine.

Was told by my PI that melanin produced by melanocyte impede polymerase activity during PCR (why he didn't tell me before i do not know...)

I found protocols online which require reisolation of DNA from cells.

 

 

Questions:

 

A) I was wondering if anybody knows how I can inhibit melanin in my already isolated DNA samples.

I did find multiple papers suggesting 'blocking' with BSA by adding it to the PCR mixture alleviates the inhibition.

I just wanted to see if anyone else had first hand experience with this.

 

B) Also, for those with experience with melanin and PCR, would melanin inhibit bisulfite conversion?

I performed Methylation specific PCR and ofcourse see very weak bands (atleast I saw something compared to wild type PCR) in both methylated and unmethylated. I assume it is because melanin inhibits polymerase, but would the conversion also be impeded?

 

Thank you.

 

 


Edited by crom80, 23 October 2013 - 10:55 AM.


#2 El Crazy Xabi

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Posted 27 October 2013 - 04:55 PM

BSA is commonly added to block inhibitors non-specifically. I use 10 ug per 20 uL reaction.

 

Diluting the sample may work too. Try different dilutions. You can combine this with the BSA modification.

 

If nothing of this works, you may need to purify the DNA or reisolate with another protocol. If you have a DNA purification kit I would try it if the BSA nor dilutions work.



#3 crom80

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Posted 08 November 2013 - 10:32 AM

BSA is commonly added to block inhibitors non-specifically. I use 10 ug per 20 uL reaction.

 

Diluting the sample may work too. Try different dilutions. You can combine this with the BSA modification.

 

If nothing of this works, you may need to purify the DNA or reisolate with another protocol. If you have a DNA purification kit I would try it if the BSA nor dilutions work.

 

Thank you for your input.

I was told by my PI that BSA addition did not work well in the past.

 

So I did a modification of a pre-existing protocol in the lab.

It was used to isolate RNA via Trizol method, briefly; "melanocytes were lysed with TRIzol reagent and lysate was incubated at 65°C for 2 minutes to inactivate melanin. Lysate was then subjected to phase extraction and RNeasy column purification."

 

For DNA, what I did was lyse the cells with TRIzol and incubate at 65°C for 2 minutes and subjected to phase extraction. Removed the aqueous layer and added ethanol to the interphase and organic layer. The mixture was then sent through the spin columns. Initial nanodrop analysis showed decent DNA concentration however when the genomic DNA was ran on 1% agarose gel I saw nothing (positive controls worked).

 

Do you have any experience in combining the two TRIzol and spin column protocols? (Qiagen DNeasy)

 

Thank you.



#4 El Crazy Xabi

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Posted 11 November 2013 - 04:00 PM

The BSA works but depends on the inhibitor and its concentration.

 

Sorry, I never used Trizol. I commonly use soil DNA extraction kits...

 

Did you check this? http://openwetware.o...for_RNA_and_DNA



#5 Trof

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Posted 20 November 2013 - 05:38 PM

Combining Trizol and Q colums is quite common, when you need better purity of RNA since Trizol can leave traces of phenol and guanidine isothiocyanate contamination. But I'm really not sure if putting the mixed organic-ethanol phase on the column is a good idea (I'm not saying it's definitelly not, but I personally wouldn't do it).

On RNA cleanup, aqueous layer with ~35% ethanol is put on column. But in the DNA prep.. you have stil organic stuff in, I wouldn't be suprised, if the column wouldn't attach DNA at all in this solution. Nanodrop can show show you the concentration of whatever absorbs around 280, like trizol.

 

 

Question is why you want to use both columns and Trizol.

If you need phenol to inactivate melanine (not sure if phenol alone can do this and what modification is required, but as the phenol is the main component of Trizol, and p-Tert-Butylphenol (whatever it may be) obviously does degrade melanine, you may try ordinary (basic)phenol/chlorophorm extraction of DNA, which makes a good quality DNA, unlike Trizol does.

 

Also if you already have your samples in Trizol, then is questionable if you really need column purity for ordinary PCR (combination is usually used for arrays and so, which are more sensitive), and if so, there are some protocols how to get rid of guanidine isothiocyanate left by Trizol. But I did some PCRs from Trizol isolation and they worked, even though I didn't dissolve it in any NaOH or whatever, just pH 8 Tris heated to 50 degs for 10 minutes, because I didn't want to bother with neutralization before PCR. It's very possible less DNA was available due to this, since it dissolves badly from Trizol, but it didn't matter.


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