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Specific primer problem

primer PGP 9.5 RT-PCR

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#1 Pipto

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Posted 28 January 2018 - 08:43 AM

Hello,

I am new to PCR. Have a problem with specific primer (guinea pig PGP 9.5) in single cell PCR. When I run a gel, I always get smears. But there is no problem with other primers from the same sample. Other colleagues used the primers before me and got good results. I thougt that there was probably contamination of primer mix, or stock primers. So I ordered it again but the problem is still there. Same sample: smears in PGP, normal bands for other primers. Also I got smears in negative water control with PGP, the same water for another primer is clear.

Do someone have any idea what could be wrong? :)

Thank you



#2 merlav

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Posted 30 January 2018 - 11:03 AM

Could you please post a photo of the gel from well to the smallest band of molecular weight marker (please add also the bp of each band)  and expected bp of amplification band.Also write the pcr reaction mix quantity and concentration of each reagent and cycles 


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#3 Pipto

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Posted 06 February 2018 - 10:08 AM

https://imgur.com/XIy59U9

 

I can not upload the picture so please clic on the link. The PGP is in the upper part. The smallest band of ladder is 100 bp and than per 100 up. Expected product size for PGP 9.5 is 129 bp. 

For reverse transcription I use Supescript III first strand syntehesis system by ThermoFisher. Than, for PCR I use Hotstart Taq polymerase KIt by Quiagen. 

The mix (for one tube):

Primer mix- 2.25 uml

Dist. mol. grade Water- 13.425 ul

10x buffer- 2 ul

MgCl2- 0.8 ul

dNTP- 0.4 ul

Taq- 0.125 ul

 

PCR: 

After an initial activation step at 95°C for 15 min, cDNAs is amplified with primers by 50 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min followed by a final extension at 72°C for 10 min.







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