emulsion PCR protocol

[Javier Diaz]

Hello all,

I talked to Sergio Peisajovich, a new PI at the UofT. He has worked with emulsion PCR's (emPCRs).

Their protocol (attached) uses also ABIL EM 90 and mineral oil. Although they used smaller scale emulsions.

I believe the following points from their paper may be of particular interest given the issues we are facing in our emPCRs:
1) Failure to add BSA or another ‘bulk’ protein to the aqueous phase will result in little or no amplification. It is speculated that the presence of a bulk protein is necessary to prevent the DNA polymerase from becoming trapped and denatured in the oil/water interface of the emulsion droplets.
2) To run a non-emulsified control along experiments. If there is amplification in the non-emulsified PCR but little no amplification in the emPCR, then the issue is the emulsification.
3) Low-quality emulsions can be identified by their appearance (they are pale and opaque rather than ‘creamy’- white) and their tendency to break down during centrifugation... we've seen this before during DNA extraction from emPCR's.

We do have Sergio very close at Systems Biology department, and he is Mariana's husband.

Best,

J.

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**********************************************************
J. Javier DIAZ-MEJIA
Postdoc at Donelly CCBR, University of Toronto.
Profr. Fritz Roth group (room 1040-B)
http://www.lusara.org/jdime
E-mail: javier [dot] diazmejia [at] gmail [dot] com
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[Joe Mellor]

I used the Williams et al protocol in the early days of BFG.  Making emPCRs in cryovials and 3x8mm stir bars is a total pain!  You don't really know if the stirring mechanism is working correctly because the solution is too opaque to see anything clearly.  

Also, since switching to vortexing (and then the IKA tubes) we don't use ABIL EM90 ...  we use ABIL WE09.  This emulsifier was used in papers from the Vogelstein and Church labs (e.g. http://www.nature.com/nmeth/journal/v3/n7/full/nmeth898.html and http://openwetware.org/wiki/Church_Lab:PoloProt)

Joe

---------------------------

Joe Mellor

Research Associate

Roth Lab |  Donnelly CCBR | University of Toronto

email: joe.m...@utoronto.ca

phone: 617-379-1589 (.us), 416-618-6246 (.ca)

skype: josephmellor

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[Javier Diaz]

OK thanks.

I was talking to Paul about the factors we believe may be influencing the emPCR:
1) MgCl2 (concentration and KCl2 instead)
2) dNTP's (old vs. new)
3) Polymerase (Stock at SLRI vs. Stock at CCBR)
4) BSA (concentration, and are we sure the BSA is active?)
5) Zymolyase (better to use fresh solution)

Suggest we test all variables in small scale non-emulsion experiments. Then see if that reproduces in the emulsified version.

J.

[Joe Mellor]

Javier,

You are apparently uninformed about a number of recent experiments that have already been done along these lines.  

There are no problems now getting fusion PCR to work in a nonemulsion reaction (with any enzyme batch of choice).   Ample evidence indicates, however, that the set of conditions optimal (or sufficient) in non-emPCR are not entirely germain to the optimal (or sufficient) set of conditions needed for efficient emPCR.    So we have an emPCR that works suboptimally compared to previous successful results, where the only change is a particular set of reagents (i.e. polymerase and dNTPs).   

I've asked Paul to perform a factorial (albeit limited) set of experiments to try to optimize enzyme and dNTPs, in addition to KCl and MgCl2., in small-scale emPCRs, on the premise that we should optimize the reaction we actual need to do.  He has done some of these experiments already, and we've already learned a bit about the positive effect of less salt.   I would characterize the current major difficulty we have as higher-than-desired amplification background, not the failure of the PCR altogether.

As far as zymo and BSA, I see no reason to optimize these further since they haven't changed and we had success with existing batches before.

Joe

---------------------------

Joe Mellor

Research Associate

Roth Lab |  Donnelly CCBR | University of Toronto

email: joe.m...@utoronto.ca

phone: 617-379-1589 (.us), 416-618-6246 (.ca)

skype: josephmellor

[Javier Diaz]

OK thanks Joe,

Yes I wasn't aware that non-emulsion PCR's were already working. That's good!!! because then is not the polymerase extract itself, but the conditions.

I mentioned the Zymolyase because in the CSHL course the instructors mention they prefer to use fresh solution each time they dissect tetrads. Apparently the activity of zymo decreases with time (stored in solution). I've looked in the internet for a reference about this, but haven't find any.

As for what you mention the "higher-than-desired amplification background" I'm wondering if this may be related with contaminant DNA from the Phusion cell extract (??) Do we have any experiment of amplification without primers? or with only one (unpaired) primer? Would a DNAse treatment of the Phusion be possible/desired?

Thanks,

J.

[Javier Diaz]

As for what you mention the "higher-than-desired amplification background" I'm wondering if this may be related with contaminant DNA from the Phusion cell extract (??) Do we have any experiment of amplification without primers? or with only one (unpaired) primer? Would a DNAse treatment of the Phusion be possible/desired?

Forgot to ask, do we see also background using the SLRI Phusion stock?

Thanks,
 

[Joe Mellor]

On Thu, Oct 18, 2012 at 6:48 PM, Javier Diaz <javier.d...@gmail.com> wrote:

As for what you mention the "higher-than-desired amplification background" I'm wondering if this may be related with contaminant DNA from the Phusion cell extract (??) Do we have any experiment of amplification without primers? or with only one (unpaired) primer? Would a DNAse treatment of the Phusion be possible/desired?

 

 

Yes, we've done the experiment of emPCR with no cells -- the background is reduced substantially but not completely.   Fatemeh is preparing a new batch of Phusion right now; the plan is to use a Benzonase-like DNAase (http://www.ribosolutionsinc.com/cyanase1.html) to help remove any DNA contamination.

Forgot to ask, do we see also background using the SLRI Phusion stock?

Yes.