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Requirement of HPLC/PAGE purified primers in Quikchange mutagenesis


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3 replies to this topic

#1 jangajarn

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Posted 05 September 2009 - 07:13 PM

I ordered primers which are just desalted (from IDT).. they are not HPLC/PAGE purified... anybody has had problems in using just desalted primers? some of my primers are ~50nt long..

#2 HomeBrew

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Posted 06 September 2009 - 05:25 AM

Primers are chemically synthesized in the 3' to 5' direction, thus the most common error is a truncation of the 5' end. The coupling efficiency of the reaction is not perfect, usually it's close to 99%. The percentage of truncated primers produced by synthesis with a 99% coupling efficiency can be estimated by (0.99 * length of primer) - 1; thus for a 50-mer, one would expect 51.5% of the primers to be full-length.

Usually, the truncated primers don't interfere with a PCR reaction, as they're all identical to the full-length primer at the 3' end, but in your case, full-length primers are essential. Since your primers are very long, and the desired mutation is in the middle (I assume), you would have a significant population of truncated primers that match the 3' base through to the 23rd or 24th 5' base, but don't include your desired mutation. These primers could function in your PCR, and cause you to generate a lot of wild-type plasmid.

If you buy desalted primers, you get the whole population synthesized -- full-length and truncated. If you elect to have your primers HPLC or PAGE purified, then a purification based of the size of the primers is being done, insuring you get the full-length primers only.

#3 jangajarn

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Posted 06 September 2009 - 01:40 PM

Primers are chemically synthesized in the 3' to 5' direction, thus the most common error is a truncation of the 5' end. The coupling efficiency of the reaction is not perfect, usually it's close to 99%. The percentage of truncated primers produced by synthesis with a 99% coupling efficiency can be estimated by (0.99 * length of primer) - 1; thus for a 50-mer, one would expect 51.5% of the primers to be full-length.

Usually, the truncated primers don't interfere with a PCR reaction, as they're all identical to the full-length primer at the 3' end, but in your case, full-length primers are essential. Since your primers are very long, and the desired mutation is in the middle (I assume), you would have a significant population of truncated primers that match the 3' base through to the 23rd or 24th 5' base, but don't include your desired mutation. These primers could function in your PCR, and cause you to generate a lot of wild-type plasmid.

If you buy desalted primers, you get the whole population synthesized -- full-length and truncated. If you elect to have your primers HPLC or PAGE purified, then a purification based of the size of the primers is being done, insuring you get the full-length primers only.


Thanks HomeBrew... I guess I'll have to order purified primers

#4 phage434

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Posted 06 September 2009 - 04:53 PM

I hardly ever use purified oligos for mutation. With 30-34 bp oligos, the mutation is around 15-17 bp in, and there is relatively low probability of fragments that short. If you are willing to sequence a few, you'll find one that works.




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