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Primer dimers

real time PCR

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#1 Sinch

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Posted 22 December 2011 - 10:49 AM

We have been running real-time PCR using a standard curve to quantify fungal DNA. The protocol and primers that we developed were working great...no primer dimers and detected DNA at 0.0001ng/ul. The sybr green master mix we using was from Eppendorf. They stopped selling this product and we switched to a sybr green master mix from 5Prime. We have not been able to reproduce our earlier results. Has anyone else had this problem? We have had the stepone real-time PCR machine calibrated. I have tried to eliminate the primer-dimers by lowering the primer concentration. Also we can not detect DNA below 0.1ng/ul. I can not figure why the protocols do not work anymore? Should I switch to a different sybr green master mix? Nothing in our protocols changed...only the provider of our master mix.

Hopefully someone can provide some insight to my problem :)

#2 Biouday

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Posted 24 December 2011 - 06:49 AM

Did you check both company products at the same time.

If you have any aliquot Eppendorf sybr green, check the same pcr reaction also with 5Prime SYBR green mix at the same time, if the you see no difference then it is problem with other pcr mix compoents like DNA, primer quality etc .

If you the see difference then check, what is difference between components of this two sybr green mix like mg con, reference dye and concentration of other pcr mix components in the mix. Most of the company wouldn't reveal the components in there mix,but give a try by calling or mailing them. Then you can understand why ur pcr is not working like before.

If nothing worked you have to optimize your condition again for this new sybr green mix but make sure they wouldn't stop selling it in near feature.

Edited by Biouday, 24 December 2011 - 06:56 AM.


#3 Sinch

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Posted 24 December 2011 - 05:20 PM

I tried to find out what was in the previous master mixes. Unfortunately I was not able to determine if the master mix changed :(

I have ordered power sybr green from AB technologies...they claim it reduces primer dimer formation.

I'll give this a try in the new year. I have exhausted all other possibilities!!!





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