Maybe a stupid PCR question & a ETBr question

Andre Hamel hamel at cc.umanitoba.ca
Sat Oct 22 18:26:37 EST 1994


Allen,

IMHO, you asked a good question ... one that I suspect begged asking for
some time. ... my personal experience from past few years is yes AND no,
regarding suitability of storing primers together (5' and 3') in same
tube, in ordder to minimize pipetting & assoc errors ... I've found that SOME
primer combinations DO NOT store well together ... thus, empirical testing
is needed before one decides to mix all primer pairs ... same holds for
storing primers together in 1x rx'n buffer, MgCl2 & dNTP ... my techs & I do
hundreds of MANY different PCRs per week (routine ddiagnostic screening
for infectious agents), and, in order to reduce pipetting & assoc erros, have
thus tested effects of storing primer pairs together with one another ...
same with storing them "ready to go, sans enzyme", in 1x rx'n buffer with
MgCl2 & dNTP ... some store VERY well in these ways, some DO NOT ...
those that do not give very reduced PCR product yields (which, in turn, are
NOT inhibitory when added to freshly mixed PCR rx'n componenets) ... I found
that for those primer combos that DO store well together, they are
best stored at -70**C ... same when storing primers together in 1x rx'n buffer
(complete with MgCl2 & dNTPs, without enzymes of course) is best done at
-70*C ... if using soon, then in frost free -20*C is okay (for up to few
weeks) ... I strongly suggest first testing effects of such storage,
compared with freshly mixed primers & rx'n mix.

I've been lucky ... the primers that I do use the most (accounts for about
1/2 of all PCR rx'ns that I do) DOES store great in 1x rx'n mix (with rx'n
buffer, MgCl2 & dNTPs ... 400 uL aliquot in -70*C). 

I'm still in the middle of testing the effects of storing 30 other primer
pairs (together in dH2O and together in 1x rx'n mix).

BTW, along same note, I've also mixed & stored MMLV-RT, RNase Inhibitor & Taq
DNA pol together (equal vol of each) ... "readdy to go" for adding to one
tube, one rx'n buffer RT-PCR ... works great ... stores well in
Stratcooler in frost free -20*C for a month so far (compared to each enz
added fresh).

Finally, does anyone have experience to share regarding storage of primers
in 1x rx'n mix that involves NH4SO4 containing rx'n buffers ... the one
that I currently use contains (for 1x buffer) 10 mM Tris, pH 9.0, 50 mM
KCl, 1.5 mM MgCl2, 0.1 % Triton X-100 ... I'm interested in testing buffer
containing 67 mM Tris pH 9, NH4SO4 & various conc of MgCl2 (1-4 mM).
cheers,



                            ********************
Andre Hamel                                    email: hamel at cc.umanitoba.ca
Manitoba Government				tel: 204/945-7630
Department of Agriculture  			FAX: 204/945-8062
Veterinary Services Branch          
Winnipeg, Manitoba, CANADA 
			  ********************


In <1994Oct17.231022.1 at cc.newcastle.edu.au> mdcabl at cc.newcastle.edu.au writes:

>Hi all,

>I realize this may be a stupid question but I have to ask it anyway.  Can I
>mix my 5' and 3' primers together, then add the combination to my pcr reaction? 
>I have historically kept them separate, labeling each stock primer tube 3' or
>5'.  I've been in a couple of labs and they seem to preserve this tradition. I
>don't see any reason a priori I shouldn't combine the tubes and save myself the
>extra hassle of keeping up with 2 stocks which are ultimately to end up in the
>same reaction tube anyway.  Maybe to carry this a bit farther, do you think I
>could add in the 10x buffer and MgCl2 and store the lot frozen?  If this is a
>stupid question or if it has been discussed before I apologize.  Any comments
>or critisms are welcome.
>	Another completly unrelated question.  What was the final verdict on
>disposal of ETBr used to stain agarose gels? Please forward me protocols if you
>can.  There's a lot of debate about it in our lab and I wasn't sure what the
>final consensus was in this group, if any. 

>Cheers,
>Allen Black 
>Dept. of Pathology
>Univ. of Newcastle




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