SUMMARY: no pcr products, Promega kits?

Marcella E Grebus mgrebus at magnus.acs.ohio-state.edu
Sat Mar 5 11:41:26 EST 1994


hey folks,

*thanks* to all those who responded to my plea for suggestions regarding
our problem with pcr [no amplification products].  we have not yet gotten
the problem figured out, as to whether it's the Promega kits or something
else.  but i'm working on it, following a number of the suggestions
you all sent me.

the following, as requested, are the responses i received via e-mail.
if anyone has any additional suggestions, i'd be elated to hear from you.

thanks again!
--marcy  <mgrebus at magnus.acs.ohio-state.edu>

---------------------------------------------------------------
Marcy, I am responding to your posting on the methods-and-reagents billboard
about Promega Taq Polymerase.  We have been using it to do simple PCR
amplifications for about a year so we have gone through the change in kits as
well.  We have done two amplifications with the new reagents and saw no
difference in yeild.  Again, the PCR we do is real simple amplification off
of Qiagen prepped DNA.  I have saved some of the old buffers in the case of
something screwy going on, perhaps you still have some kicking around your
lab.  At least you can see if it is a buffer problem.  Good luck -
Brian Zeiler
brianz at lifesci.microbio.ucla.edu
---------------------------------------------------------

From: <tangent at unity.ncsu.edu>

Marcella,

A brief reply. We use promega kits a LOT, and know that one kit can differ
considerably from another. For that reason we keep kits separate and record lot

numbers as routinely as any other component of the reaction. We've had some
extremely crappy 10X kits from promega that we've simply discarded. We've also
done the experiments to compare buffers: different lot numbers can and do
give different results - another unwanted variable. Usually the 10X is the 
cause of variation, rather than magnesium.
What is the lot number of your new kit - we may have a similar lot with equally
bad results, or alternatively we could eliminate that from your list of
possibilities.

A tip - try 1 mg/ml non-acetylated BSA (from NEB) - it works extremely well for
improving the level of amplification and repeatibility.

Phillip Wilcox
Forest Biotech
NCSU.
----------------------------------------------------------
Have you contacted tech/sales rep at Promega regarding your faulty Taq?

Also, could you mention the lot number? Sounds like a faulty batch of Taq.

Was it send on dry ice?

regards,
                            ********************
Andre Hamel                                    email: hamel at cc.umanitoba.ca
Manitoba Government Veterinary Services          lab tel.: (204) 945-7630
545 University Crescent,                              FAX: (204) 945-8062
Winnipeg, Manitoba,
CANADA   R3T 5S6            ********************
--------------------------------------------------------------
From: "FrohlichM" <frohlichm at starbase1.caltech.edu>

Marcy,

I had similar unexplained total failure with a kit from Perkin-Elmer a couple
years ago, that turned out to be caused by the MgCl2-  The actual
concentration of MgCl2 IN SOLUTION was about 1/4 what it should have been. 
After I found the problem I asked their rep, who said the MgCl2 precipitates
on freezing and may not redissolve when you thaw the tube, even if the tube
stays thawed for a long time.  Mixing my own MgCl2, which I never freeze,
solved the problem completely.  Of all the reagents this is the simplest, so
I wasted a lot of time trying other manipulations before I checked the MgCl2.
 I expect the same problem could also happen with solutions from other
manufacturers.

Good Luck,

Michael Frohlich
---------------------------------------------------------------
From: Louis van de Zande <ZANDELPW at biol.rug.nl>

Hi,
Well first try some of the old batch (or has that been used totally), 
Then try some Taq from the nextdoor lab and finally contact Promega 
and confront them with the problem.

Yes, I know of "The simple fools guide to PCR" which has some good
advice aln also a list of useful primers. As the titel suggests, it
is a lucid protocol, but I find it very useful.
If you are interested, give me the address and I will send you a
photocopy.
Good luck
L.van de Zande
Dept. of Genetics
University of Groningen
P.O. Box 14
9750 AA Haren
The Netherlands
Tel. +31 50 632126
Fax. +31 50 632348
------------------------------------------------------------
From: jgraham at bronze.ucs.indiana.edu (the End)

Sure,

Make your 10X salts solution and make it at 5X, and don't freeze it,
but filter sterilze and store at room temperature.

200 mM KCl
50 mM Tris-Cl (8.3)
7.5 mM MgCl
0.05% gelatin

We also add 0.05% NP-40 deteregent and 5% Acetamide (10/100 ul of 50%) to
all final reactions. I add MgCl to 2.5 mM when using plasmid targets.

If that fails, reconstitute a new batch of dNTPs

If that fails, purchase some new Taq polymerase (fully licensed for PCR of
course ;)

Now throw away the silly "PCR Kit".

It is essential that you be able to trouble-shoot the components of the
reaction yourself (at this stage), as later on that skill will be
necessary in your own experiments.

Best wishes,

Jim
J. Graham
----------------------------------------------------------
From: Stephen T Simpson <simpsst at mail.auburn.edu>

Marcy,
     Forgive me if I send this note the wrong way, but I believe you 
asked about possible causes for PCR reactions dying out.  Well, I have 
always found this sensitive reaction to change by some unknown force.  I 
try to do everything the same (even the PCR chant and dance) each time 
but still get varying results.  It must be my poor method, because people 
are publishing papers that include quantitative PCR (are you as skeptical 
as I?).  
     But back to your question, I know you said  your reaction 
buffers were the same, but I understand that Invitrogen (I think) has
created a PCR optimizer kit.  It came out after I was done with PCR, but 
a girl in my lab used it and said it worked well.  It basicly just comes 
with a set of different buffers with varying Mg, salts, etc.  Though I 
hope you find the problem (like the old dNTPs, that was a good thought), 
I just thought I'd share that with you in case you have to compleatly 
reconstruct the reactions.  
     Good luck...  and post or mail me a summary of responses, please.

Stephen
-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-
Stephen T. Simpson                 Scott-Ritchey Research Center 
(205) 844-5951                     College of Veterinary Medicine 
simpsst at mail.auburn.edu            Auburn University, AL 36892



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