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-   -   Is a purification step between cDNA generation and library construction necessary? (http://seqanswers.com/forums/showthread.php?t=58566)

kobeho24 05-15-2015 02:11 AM

Is a purification step between cDNA generation and library construction necessary?
 
Hi all,
Recently, I was wondering that is it necessary to perform a purification step after cDNA generation and prior to library construction? Because I am gonna do the cDNA generation in a very tiny volume. The library construction can be either ligation-based or tagmentation-based.
Best!
Gary:)

Simone78 05-26-2015 12:48 PM

Quote:

Originally Posted by kobeho24 (Post 172549)
Hi all,
Recently, I was wondering that is it necessary to perform a purification step after cDNA generation and prior to library construction? Because I am gonna do the cDNA generation in a very tiny volume. The library construction can be either ligation-based or tagmentation-based.
Best!
Gary:)

itīs not necessary at all! Just dilute the PCR product to reduce the amount of salt, take an aliquot for ligation/tagmentation and go on. I tried it myself and it works quite well. I never compared the efficiency with a "standard" protocol, though. Based on what I see on the Bioanalyzer (havenīt sequenced any sample yet) I would be surprised to find out that the difference is so dramatic.
good luck!

Chipper 05-26-2015 10:38 PM

No, you can do end repair after the 2nd strand reaction without purification.

cmbetts 05-27-2015 10:52 AM

I believe the main reason you typically see cleanup between 2nd strand and end repair is that the 2nd strand buffer for most systems has ammonium sulphate in it, and the T4 PNK used in end repair is potently inhibited by ammonium. It might work out if the reaction is diluted enough that the ammonium is a nonfactor.
Ex, NEBNext
20 mM Tris-HCl
12 mM (NH4)2SO4
5 mM MgCl2
0.16 mM β-NAD
0.19 mM dNTPs each
pH 7.4 @ 25°C

nucacidhunter 05-27-2015 09:27 PM

I think by cleaning up cDNA uniformity and consistency among samples and batches will be increased. Non-cleaned cDNA would have various amounts of left over oligos and salts which may affect library quality.


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