i am having some problems with my PCR and i hope some of you
might provide me some insight:
i have to amplify a 1200bp product from bacterial genomic DNA
with primers designed from a closely related organism. the first
round of PCR with 45 degree C gives a faint 'expected' band along
many non specifics. when i used a 5ul of that PCR reaction as a
template for my next PCR with 50 C annelaing -i got a good
"expected brand' along with some non specifics. there was no
i gel purified the expected band and used the gel extracted
DNA as template to reamplify it- that also amplified the expected
band but there was a internal smaller band amplification.
so, at this stage i cannot use my pcr product as such for TA
cloning - i had to gel purify the band- so i scaled up the reaction to
get more expected band.
then the trouble started- from then on- always when i try to amplify
my1200 band from PCR product as template or gel cut band as
template-, when i run my gel-the whole lane would be a big long
smear along with some 1200bp amplification. this whole smear
bothers me and this is happeneing only for some time now and i
used both taq polymerase and also stratagene Easy A high fidelity
enzyme- the second enzyme is giving more smears
i know this post is long- but i am desperate bcos i have tried many
optimizations and a seemingly simple step is blocking the whole
i eagerly wait for your suggestions.
thanks for your time.
Historians believe that in newspost
<[Only registered users see links. ].mrc.ac.u k> on Thu, 9 Oct 2003, [Only registered users see links. ] penned the following literary masterpiece:
Why can't you use it?
Two products so 50:50 chance of getting the right one if you picked a
white TA clone. So screen 20 whites with M13 universal/reverse primers
or your 'specific' primers and sequence the clone with the right size
insert. For all you know the small band could be an internal fragment
PCR from that 1200bp.
Every time you re-amplify you will generate more and more background due
to excess primers, false priming and therefore product you couldn't see
from the earlier PCR etc. etc. Basically don't do it! TA what you had
and you may well be OK.
I love deadlines. I especially like the whooshing noise they make as
they go flying by.
it's hard to tell what really happens unless you find it's DNAse or
microbes what causes the smear. all you can do is carefully optimize your
PCR if it makes sense wasting the time and the money.
do you have the possibiliy to run a gradient pcr with different annealing
temperatures or at least a touch down pcr? this might eliminate the
parasite bands. for re-amplification, try to increase the annealing temp to
at least 50 or 55 degC - how long is your primer and how degenerate is it
(probably)? in the second round, the primers are specific for the ends you
have generated in your first round.
why do you need the re-amplification step at all? the more cycles you have
in total the higher is the risk of mutagenesis by error-prone-ness of your
enzyme, esp when you are using Taq. just cut out your band of interest and
clone it. You also could use the band for direct sequencing it with both of
your primers. just to be sure that it is what you want. murphy's law says
that the band of the right size rarely is what you actually wanted to
amplify :-( - bad multiple experience of my own.
you also might try adaptor primers instead and clone into a RE cut and
dephosphorlated vector. EcoRI, XhoI, HidIII, BamHI are enzymes often
suitable for this purpose, it only matters that your fragment is not cut by
them (test that before or simply find shorter inserts.
At 17:36 09.10.2003 +0100, [Only registered users see links. ] wrote:
In article <[Only registered users see links. ].mrc.ac.uk> , [Only registered users see links. ] wrote:
Firstly I have successfully TA cloned bands I could barely see on the
gel. The trick is to have a high % recovery scheme, I get my bands out
by running them into low melt wells and then using agarase enzymes to
digest the agarose. The resulting long chain soluble carbohydrates seem
to act just like glycogen, funnily enough, and enable pull down of small
amounts during precipitation steps. If you use spin column methods you
run into problems, not least with minimum elution volumes.
Secondly, if you are doing multiple rounds of pcr you really need nested
primers to ensure you are reselecting only valid sequences. Nesting can
be difficult to achieve, but if you can only nest one end that is better
School of Life Sciences, University of Dundee, Scotland
To assume that I speak for the University of Dundee is to be deluded.
Reverse the Spam and remove to email me.
that is a very good thought.
Are you aware of any source that summarizes all these PCR woes and teaches
how to overcome them? Maybe a book or website that does not only explain
the theory of PCR itself but emphasizes the theories of the errors that may
(and will) occur?
hi, I have a very similar problem, the only difference is that I have to produce longer products (up to 6.5 kb), and I have to make several micrograms with PCR. I have three fragments, call them A, B and C. With my test-template, all of them worked (genomic DNA). Later I tried it with other samples, some worked, some not. After a lot of failures, I don´t know what happened, fragment C amplified from ALL of my templates, but A and B NOT (usully 2-4 out of 8-10 is working though in A and B, not always the same samples for the two fragment). So what I conclude:
- cannot be the template, because it worked for fragment C
- cannot be the primer, bceause it works in some sample in each run
- cannot be the mix (dNTP, enzyme whatever), because samples from the same mastermix behave differently.
What I can think of: I´m using too much of template. I´ll try to reduce it, and combine it with touchdown. But I really have to get some result very soon so please if you have any idea (and not cloning) I´d apprectaite it very much!
When I have smearing, I'll try the following first, I'll tweak the Mg conc in the PCR rxn. Also ensure that extension time is not too long (check the extension rate of your DNA polymerase). I'll prepare my rxn according to order... dH2O, buffer, Mg, dNTP, primers, template and DNA polymerase being added last. If it's not a Hot Start DNA polymerase, the rxn should be prepared on ice at all time.