Primer purity

[mansipan]

After designing a primer there are different purity grades available according to the purification protocols adopted like, desalting, reverse phase cartridge purification, RP-HPLC, AX-HPLC, PAGE and Gel filtration. my question is how much purity or which purification procedure of primers is good enough for amplifying a gene for cloning purpose.

[CharonY]

For PCRs desalting is usually enough.

[mansipan]

For PCR its ok i know but i want to know for further cloning, whether one should go for purification of primers. Is it so that, desalted primer PCR amplified genes give problem in cloning.

 

Reference: Invitrogen

Cartridge, HPLC, and PAGE-purified oligos are best for the greatest efficiency. Since oligos are synthesized 3´ to 5´, incomplete oligos (n-x oligos) will be missing the 5´ sequence. It is important to use full-length oligos that have the 5´ sequence present, otherwise there will be a population of PCR products missing the sequence intended to be installed before PCR"

[mansipan]

Please reply CharonY

[CharonY]

it is usually a non-issue as the efficiency of the whole cloning experiments tends to be way lower than that of the mismatch rate. You have a slightly higher chance of creating some false products, but depending on how the designers are designed and how the cloning is going to proceed you will end up with the vast majority of the correct product for cloning. Since you have to run controls anyway, the probability of PCR purity being the culprit for failed cloning is very low.

There are exceptions however, e.g. if you do some tricky amplifications with very low yields. Salts or impurities themselves are no issue at all as you do a cleanup post-PCR, anyway.

[mansipan]

it is usually a non-issue as the efficiency of the whole cloning experiments tends to be way lower than that of the mismatch rate. You have a slightly higher chance of creating some false products, but depending on how the designers are designed and how the cloning is going to proceed you will end up with the vast majority of the correct product for cloning. Since you have to run controls anyway, the probability of PCR purity being the culprit for failed cloning is very low.

There are exceptions however, e.g. if you do some tricky amplifications with very low yields. Salts or impurities themselves are no issue at all as you do a cleanup post-PCR,

Thanku very much