PCR of larger DNA Fragments: summary

nigel at codon3.berkeley.edu nigel at codon3.berkeley.edu
Thu Nov 21 11:28:13 EST 1991


Hi folks,

I recently posted here, looking for help with my PCR optimizations.  I am
trying to amplify a 5kb transposon out of maize.  The protocol I stared with
was based on a "vector PCR" protocol published in October's BioTechniques [p446-452]:
>                - 100uL rxn vol.
>                - 200uM dNTP
>                - 1.5mM MgCl2
>                - 50mM KCl
>                - 10mM Tris  [pH 8.3]
>                - 0.01% gelatin
>                - 25 pMoles each primer
>                - 2.5 U AmpliTaq
>                - 1-10 ng template
>
>        - 2 min @ 94 C; 2 min @ 52 C; 5 min @ 74 C   for 30 rounds.

The following are some of the suggestions that I recieved:

JUST_W at rz.uni-ulm.dbp.de <Walter JUST> writes:

>Please refer to Nucl.Acids Res. _18/4_, p.1079 (1990). A colleague at our
>university was using gp32 for enhancing PCR of products up to 6.5 kb.
>gp32 is available from Pharmacia Biosystems.

I haven't tried it yet...it's on order (and I will let you know if it helps).

JBISHOP at POMONA.CLAREMONT.EDU <J. Bishop> writes:
                    
>                       .....most important is that you get a working
>primary amplification of your template.  I have found that it often
>works to set your first cycle of denaturing, annealing, and amplification
>for twice as long as the others, i.e. 4 minutes at 94, 4 minutes at 52, and
>10 minutes at 72.  Subsequent cycles can then be performed at the original
>times.  I might suggest that you lengthen time period allowed for amplification
>Five minutes is not necessarily enough time to lengthen a 5 kb fragment. Third, 
>you did not state how long your primers were, but you might want to try using
>a lower annealing temperature.  For 10mers I use 42 degrees.
>These are just basic places to start, but they have worked for me in the past.
>
> oh, yeah, you might want to include a ten minute run off at the end, though
>I don't suspect that will help if you aren't getting any amplification at all.

I haven't tried this..and at the moment I don't intend to, the times just seem
WAY too long.  The run off at the end sounds good, particularly if I was 
intending to clone the product.

snella at biotek.ifas.ufl.edu <Liz Snella> writes:

>Do your primers work when you try to amplify smaller regions?

Yes!  Good question though, I was really worried about that for a while [o/n].

>If you have any old 32P hanging around, try filling in the ends of the products
>with label.  The 8 KB fragment that I mentioned is not visible on Ethidium gels
>but is visible if you endlabel.  Then run the labled fragments on a agarose gel
>dry it and expose it. Even with month old label it only takes an overnight
>exposure.

I blotted and did a southern instead...I had hot probe already. [nothing!!]

>Try doing a "hot start", before you place the tubes in the machine have it up
>to 94 already and pause for several minutes before you start the first cycle.  

Good idea: but you need to leave either the Taq or the template out untill the
reactions are at 94oC for it to be effective (I'm sure this IS what Liz meant.)

The weird thing is that no-one suggested mucking with the [MgCl2] (too obvious?)
I dropped it 3 orders of magnitude (1.5uM) and started to see amplification.

NBR at AC.DAL.CA <Bruce Ramsey> (in an unrelated posting) wrote:

>...... I've on occasion obtained PCR products
>which consist of lambda sequences entirely - due
>to priming from the 3' seven or ten bases of a 17-mer
>and 20-mer respectively.  While the front end of these
>primers were dissimilar to any lambda sequence, the 
>downstream ends were perfect matches.
>
>In other words, we should definitely check the 3'
>half in particular against the vector sequences.

I agree 500%!  I have 3 or four smaller bands ( more intense than the product
I'm actually after) that ARE due to a 9bp and two 7bp stretches of homology
at the 3' end of my primer!  Very poor design on my part.

I still have a ways to go before I get this particular amplification optimized
and I'm more than willing to share my experiences with those who are interested
(I'll post another summary if I get more that a couple of requests).

Thanx for your help!
-n

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