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Problems regarding amplification of my gene


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#1 rubhadevi

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Posted 11 October 2014 - 12:25 AM

Hi. My Product size is 405bp. I have isolated RNA from mouse epididymis and i converted RNA to cDNA by using reverse transcriptese (Applied Biosystems). from the first time, i got amplification of my gene (405bp). After that i could n't found reproducibility. I dont know the exact problem. Then i had changed two more set primer from different company. Till now i couldn't solve my problem. Please suggest me to troubleshoot go get amplification. here i attached my gel picture.


Edited by rubhadevi, 11 October 2014 - 01:05 AM.


#2 Tabaluga

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Posted 11 October 2014 - 01:11 AM

Hi, I cant see any gel picture attached...

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#3 Ameya P

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Posted 11 October 2014 - 02:43 AM

(Probably, there are restrictions on attaching files to her post)

 

Anyways, primers are unlikely to be your problem. If you managed to get the PCR to work one time, you should try and replicate its result rather than introducing something new. 

 

Probably, you had pipetted Mg or dNTPs twice or your template did not have sufficient cDNA for the amplification to occur again. Can you do any other PCR on the cDNA to show that it is working well? 


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#4 rubhadevi

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Posted 12 October 2014 - 02:13 AM

I am using master mix from takara. Concentration of cDNA around 800ng/m. I had tried with different gradient. I want to know if the bases of sequences more than 30 will make problem? and query coverage of sequences through blast around 46%.






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