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
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
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.
 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
(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).
 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.
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.
William Moens Ph.D.
IHE - Lab Biosafety & Biotechnology
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.
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