summary: re-amplification of PCR product using the original

BHWFDUNG at CPHKVX.CPHK.HK BHWFDUNG at CPHKVX.CPHK.HK
Fri Aug 26 04:33:33 EST 1994


Hi netters,

Thank you for all those kindly people in the net for giving me some 
suggestion on re-amplification of PCR product using the original PCR 
primers.

This is a summary of the messages I received and I think it will be useful 
for the other people doing the similiar work.

I have tried to dilute the first PCR reaction mixture from 10^4 to 10^7 and 
then re-amplified it using the same primer pairs. But, no luck! I still 
got smear for all the dilution.

There are several thing I want to state clear. 

[1] I am doing a touchdown PCR using ONE of the degenerate primers as 
forward primer designed from a highly conserved region and another ONE 
of the degenerate primers as reverse primer which are from another highly 
conserved region.

(The idea is based on a paper in Biotechniques 94, that touchdown PCR 
can be used for amplification to overcome the problem of mismatched base 
in primers due to codon degenercy).
 
[2] The condition using for touchdown PCR is quite stringent, from 70 C to 
60 C, and then followed by a normal 30 cycle amplification with annealing 
temp at 60 C. I got three distinct bands. But the yield is not good so I 
decide to do re-amplification.

I think if I purified my interested band after gel electrophoresis and 
then dilute it to 10^6 or 10^8, the situation may improve because my 
first PCR give multiple bands already.

Thanks again!
Nelson
bhwfdung at cphkvx.cphk.hk 
=========================================================================
From: Moens William <wmoens at rc4.vub.ac.be>

We do exactly what you wish to do without problems for now 5 years.
Take 1 microliter of the first PCR reaction, dilute 1/100 - 1/1000 in 
autoclaved water, add 1 microliter to the next PCR mixes. The smear 
indeed occurs if too much previous mix is added. Dilute till it dissapears.
This works for fragments between 200bp up to 1.8kb, with consensus 
primers amplifying a family of genes (then you have to get a smear) or a 
library of cDNA (see Boehringer's kit for example). The smear problem 
might originate from bizarre primers by-products of the first reaction 
but we did not explore the mechanism.

Tell me whether this helps.
Cheers

William Moens Ph.D.
IHE - Lab Biosafety & Biotechnology
Brussels Belgium
=========================================================================

From: sjscharf at netcom.com (Stephen Scharf)

try 10^5 or 10^6 dilution of your PCR target, see if that works, usually
a smear means you have too much in your PCR.

========================================================================

From: acer <forrest at florey.biosci.uq.oz.au>

How were the outside primers designed? are they degenerate or consensus 
primers based on related organisms? If so once the primers have been 
incorporated ( during the first 3-5 rounds of amplification you can bump 
up the annealing temperature. I'd probably work on the first PCR more than
trying to reamplify, it's more likely of giving you the result you're after
in a shorter period.

But if you have to reamplify, 1stly run out your sample on a gel to give, 
the best separation you can. Then you can either try amplifying from a 
small plug of gel (using a pippette tip) or you can purify it using 
something like geneclean.
To minimise non-specific amplificationyou can try massive dilutions ( try a 
dilution series to at least 1 in 10000). It may also be worth while trying a 
heat soak step before adding your enzyme ( heat at 94 for 30min ).

I hope this helps. I did a lot of this type of stuff during my honours year. 
Reamplification is fine if you're going to clone the product, but if you 
want to sequence the PCR product directly ( especially with cycle 
sequencing) the background smear ( no matter how faint ) will interfere 
with the sequencing ladder as both the smear and the product of interest 
contain the primers you, have amplified and hope to use for sequencing with.

============================================================================

From: Big Nose <andy.law at afrc.ac.uk> (Tel \(0\)31 440 2726)

What did your original product look like ( before you reamplified it? ). 
Are the primers specific for your sequence (are there a family of gene 
products that may be interfering). 
Are the primers designed in one species and used in another?

You could tinker around with the conditions for PCR (increasing temperature 
is one possibility), but you may be there for ever doing that.

If you have 0.1ng of purified product, I would suggest that you clone the 
original product into a T-tailed vector then grow a few colonies up and 
either PCR from the colony plasmid DNA (that way you know that your template
is homogeneous) or sequence a few of them. If you sequence them, you also 
get the information you need to design internal primers.

============================================================================




More information about the Methods mailing list