PCR scaling up doesn't work - (Apr/07/2005 )

I used one set of primers to do 50ul PCR reaction, it worked well. But now i ordered the same set of primers again and tried to do 100ul PCR reaction. There were some PCR product, but very light. Could anyone suggest what is the problem maybe?

Thank you for any of the idea.

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Have you tried a 50ul PCR with the new set of primers? This way you could understand if the problem is with the primers or the PCR conditions.
Have you doubled all reagents, DNA included, in the 100ul reaction?
W.

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I think you want as much product as possible so you preformed a PCR with 100 ul.
Why not prepare two tubes, each contains a 50 ul reaction mixture? since your 50 ul system is quite optimal already.

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did you use the same amount of primer in your 100ul reaction as your 50ul reaction? if you did, that would explain things, you would have to double all the amounts of primer and starting template as well.

Nick

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I doubled all of the things when doing 100ul PCR. But it did not work very well.

I did 50ul again with new stock of primers. I found that the 50ul did not work very well either. Is there any problem with new stock of primers or some other reasons?

thanks very much for lots of people reply.

Freshman

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hi
try increase the number of cycles according with bullfrog recommend.
is your template the same in two experiments, or did you re-prepare it? (by writting that i wonder about the purity of your eventually second prep...)

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Just check if the dilution of the primers stock you've done it's ok. Otherwise, the new primers are the problem. Do you have any rest of the old aliquots, so to compare the two in the same reaction conditions?
This happened to me too, poor quality of oligos...
Cheers
W.

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I often find with my students, or myself, that making a FRESH primer mix solves the most common reason that reactions suddenly start failing: primers degrade.

When you get new primers, typically dried down, hydrate them in TE (10 mM Tris, 1 mM EDTA, pH 8). Keep that stock in TE, so it will survive many freeze thaws when you make up primer mixes.

If you make your primer mixes, for setting up reactions, in water; they may or may not work a few more times. Non-buffered primers start to slowwwwwly degrade. You can use 1/2 x TE or even 10 mM Tris pH 8 for mixing up your primer solutions from the original frozen stock tube.

If you use invitrogen or ITDDNA, they tell you how many nmol are in your tube. I find those measurements are are trustworthy. If you did your own concentrations using the UV spec, double check by re-spec'ing them.

have fun,

http://ken.mitton.com
Dr. Mitton

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