direct PCR amplification using library
Sergey V. Orlov
Serge at serge.stud.pu.ru
Mon Aug 28 16:46:20 EST 1995
> I am looking for a detailed protocol for dirct PCR using cDNA library
> as the DNA source. I am using denatured (boiling for 5 min) 1ul phage stock
> (approx. 100,000 pfu) in PCR reaction. Unfortunately I am not getting
> any amplification product. Only one of the primers is specific for the
> gene of my interest, another is T7 or T3 primer.
> Thanks in advance
Do you sure that your pair of primers is compatible (i.e. thees primers
have similar annealing temperatures, don't form dimers etc.) ? Another
reason is that your reaction conditions don't optimum. Recently, list of
parameters affecting on PCR was published in this newsgroop by Bettye
Smith. I'm coping his message below. Try vary any parameters.
E-mail: serge at serge.stud.pu.ru
In article <1995Jul28.184151.1540 at newsserver.rrzn.uni-hannover.de>,
bmay at 126.96.36.199 (bmay) wrote:
> I'm trying to amplify 2 parts of a single copy gene from human DNA prepared from buffy
> coat with Qiagens QIAamp Blood Kit. I'm using 2 pairs of primers already used in published
> literature (published annealing temp. 65/68) designed to yield fragments of ~190 and
> ~250 bp. 100l reaction volumes contain 200 M dNTPs each, 1.5 mM MgCL2, primers 50
> pmoles each, 1 g DNA and 2 u cloned Taq (USB) in buffer supplied. Cycles consist of 1 min
> denaturation 95, 1 min anealing 58 and 45s extension 72 with 5 min initial denaturation
> and final extension in a Crocodile II cycler from Appligene.
> MY PROBLEM ARE INCONSISTENT AMPLIFICATION RESULTS: EITHER I GET NOTHING (LOOKS LIKE A
> PERFECT NEGATIVE CONTROL) OR I GET SINGLE BANDS BUT I CAN'T REPRODUCE THEM THE
> OTHER DAY. SOME DNAS YIELD SINGLE BANDS WITH ONE PRIMER PAIR AND NOTHING WITH THE
> OTHER AND VICE VERSA. I'VE ALSO FAILED TO REAMPLIFY A SINGLE BAND I ONCE GOT!
> Any hints, suggestions or comments wellcome !!!
> Thanks a lot in advance.
> Bernhard Mayr
> Dept. Clinical Endocrinology
> Medizinische Hochschule Hannover
> e-mail: ndxdbmay at rrzn-user.uni-hannover.de
Dear Dr. Mayr,
Many variables can affect the success of PCR, and each element has critical
parameters that need to be optimized. The concentrations of the components
of the reaction are important, and have to be optimized in individual
settings, dependent on the thermocycler used, pipetting, type and size of
DNA, purity of DNA etc. Multiplex PCR is even trickier, I think. IÕve
included some suggestions, based on personal experience, that I hope will
1. Try varying the concentration of dNTPs. IÕve found that high
concentrations of dNTPs can affect PCR conditions. When dNTP is 200 uM,
there is enough to synthesize 12.5 ug of DNA when half the dNTPs are
incorporated. dNTP chelate Mg, and thus change the effective optimal Mg
concentration. Additionally, >200 uM dNTP (each) increase the error rate of
the polymerase, and millimolar concentrations can inhibit Taq. Current
Protocols recommends preparing the dNTPs in 10 mM Tris/ 1 mM EDTA pH
7.4-7.5, instead of neutralization with sodium hydroxide. Remember though
that EDTA will chelate Mg, and this should be taken into consideration when
optimizing the reaction conditions.
2. Vary the DNA concentration. Some of our customers have found that less
template is needed in PCR reactions, because QIAamp purified DNA is very
clean. One customer (in an HLA lab) found that she needed to decrease the
template concentration ten fold! Start with 100-200 ng, and see if that
helps. 100 ng of genomic DNA is sufficient to detect a PCR product from a
single-copy mammalian gene. Using too much template is not advisable when
optimizing MgCl2, or other parameters, as it may obscure differences in
3. The MgCl2 concentration is also an important variable. Current protocols
suggest settting up a series of reactions for MgCl2 optimization from 0 to
4. Avoid using DEPC treated reagents as DEPC is an inhibitor of PCR.
5. Use of high quality reagents will often avoid nuclease contamination.
6. G-C rich primer sequence can often lead to secondary structure or
7. Other components: Some investigators have found substituting NaCl for
KCl and/or altering the pH to be useful. Others have altered the gelatin
concentration (I omitted it completely, without deleterious effects). Some
have added DMSO (3-10%, or various concentrations of glycerol, especially
if the DNA has a high G-C content.
8. Since multiplex PCR can be difficult, try each primer pair individually
first, before adding both primer pairs in the reaction. The primer pairs
could be interfering with each other, and thus by optimizing the conditions
separately first, may alleviate some initial problems.
9. Finally, optimizing annealing/ denaturation temperatures and times will
improve PCR efficiency.
I hope this helps. If you have any questions, please feel free to contact
our Technical Services department at (0) 2103-892-240 (Germany).
Bettye Smith, Ph.D.
(818) 718 9870
Temporary Email Address qiagen at kaiwan.com
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