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Genotyping conditioning-where to start?


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#1 SMN

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Posted 25 August 2009 - 04:12 PM

I've got a pair of KO mice from a collaborator, however, I can see any band when I do the genotyping using WT mice with the primer sequence provided from them. Even I follow their PCR set up and use the reagents from same company, I still can't see any.

My collaborator keeps tell me that it's a "non-efficient" PCR reaction as the PCR product is about 1kb. More DNA should be added and amplification cycle should increase. However, increasing DNA (Prepared from Qiagen kit, max 300ng @ rx) and cycles (max 40) don't help.

I have the primers prepared from 2 companies, so it should not be the problem of primers.

I think I may need to optimize the PCR condition myself. Where should I start from? Should I start from the concentration of reagents first (e.g. MgCl2, dNTP, primers)?

For Taq, I've tried New England Biolab and Roche, both are newly ordered and working good on other genotyping.

It has been frustrating for a long time and I do need some experts to help.

#2 k_undertoe

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Posted 29 October 2009 - 12:48 PM

I've got a pair of KO mice from a collaborator, however, I can see any band when I do the genotyping using WT mice with the primer sequence provided from them. Even I follow their PCR set up and use the reagents from same company, I still can't see any.



When you isolate genomic DNA, do you make sure to use the Qiagen kit's elution buffer (withOUT EDTA); because if you use TE buffer instead, it can inhibit downstream amplification by interfering with the enzymes/salt optimal concentrations.

Is you DNA clean? (spec reading of 260/280 and 230/260 are good). You may need to do an ethanol/NaAcetate precipitation to eliminate excessive salts, which can again interfere with the optimal enzyme/salt concentrations for your PCR reagents. Too much salt won't allow your primers to anneal correctly; it stabilizes the DNA too much and they won't bind to your primers. I'll get back to salts later...

My collaborator keeps tell me that it's a "non-efficient" PCR reaction as the PCR product is about 1kb. More DNA should be added and amplification cycle should increase. However, increasing DNA (Prepared from Qiagen kit, max 300ng @ rx) and cycles (max 40) don't help.


Actually, as counterintuitive as it may seem at first, adding more template can actually really lessen your success of an outcome. Adding too much template interferes with the reaction by either binding all the primers so that you don't get your doubling of target each cycle, competing for “space” with the big polymerase enzyme in the reaction mix and making amplification inefficient and non-linear; so more is less, in a sense… The amount of 'extraneous' or non-target DNA in the sample must be carefully balanced with the target for reaction volume space along with the required amounts of primer to get the job done. I've heard that for genomic DNA templates (I'm guessing that is what you are using), between 0.1-2pg per 50 microliter reaction should be the operating limits. Another possibility is that your DNA prep contains too many inhibitors and by lowering the concentration you can decrease them and perhaps get product, but if your target is not very abundant this may not get results as the concentration becomes limiting.

Since your collaborator has gotten their PCR to work using the specified reaction conditions (primers, cycle, reagents) I would try to run a serial dilution of your template to see if adjusting that amount can get you anywhere. If you've already tweaked the starting template concentrations, see below for more suggestions.

I think I may need to optimize the PCR condition myself. Where should I start from? Should I start from the concentration of reagents first (e.g. MgCl2, dNTP, primers)?


There are many opinions as to how you should try and optimize your reaction. First of all, I would make sure you are within the recommended parameters set forth by the brand you use for their specific product regarding dNTP, primer, MgCl2 etc. concentrations. Make sure your starting template doesn't have too much extraneous salt or other contaminants.

When I am trying to optimize a PCR usingsome else's primers, I take an aggressive approach where I adjust a few parameters at once rather than to try and tweak them one at a time. When I have a 'tried and true' primer pair but I'm not getting product, I run 2 different primer concentrations, (and this might help with reconciling the non-target DNA vs your target template,) and also decrease the annealing temperature in step of 1 degree C with an increase in the extension time of 30 seconds. Hopefully this will increase the availibility of my primers to reach my target and do their magic, and will compensate for a stabilizing too much salt imbalance to some extent. The caveat for this is that temp. lowering and ext. time incr. can cause primers to anneal with less stringency and give non-specific amlicons.

Alternatively, there are many products on the market that claim to 'enhance' or 'guarantee' a PCR to work by adding stabilizers and other ingredients to make a more robust mastermix that will try and force your reaction toward a product (e.g. FailSafe PCR Mix {no affiliation} or you can add your own stabilizers such as DMSO). If you don't want to try those ($$$!) you can, as you say, try to adjust the components for your mastemix yourself; for that I would direct you to some resources that can give you a set by step plan so you aren't just changing things willy-nilly and get confused. For that I would check out the optimization recommendations for your enzyme manufacturers (they all have one or more) on how to use their mix and get it to work. Just once more, I'll emphasize that of critical importance for your reaction is salts. Salts don't just work at the DNA-to-primer interface, they can also have a negative influence on the pol enzyme itself, inhibiting its efficiency. Reaction components (dNTPs, primers, template) can reduce Mg2+ availibility to the pol, which needs it to work, but if you already have a salty mix, adding Mg2+ be more available, but the other salts stabilize the mix too much for it to have a benefit. Anyhow, a fairly decent, informative resource is the Yale PCR page, and contains info about adjuvants and concentrations of separate reagents in distinct chapter/section areas, but if you want a step by step guide, like I said I'd go to either the pages for your products or for your PCR machine, ABI and BioRad have huge repositories of optimization protocols.

Hope this helps a little, good luck and good PCR karma to you!




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