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problems regarding amplifying a 1.7 kb mRNA seq


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#1 rubhadevi

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Posted 17 March 2014 - 08:24 PM

i want to clone 1.7 kb mRNA seq. here i followed two set of primers (from published papers). i was tried to convert RNA from cDNA by using random hexamers. i didn't get amplification of primers from my gene of interest. i dont know the problem exactly with cDNA quality or primers and also i used internal control GAPDH for to check the cDNA. i got amplication in internal control. i was tried different gradient, different concentration of cDNA and also i used pcr additives. plz find a problem and give me a idea to do amplify my gene.here i attached some details about pcr reactions ,

 

maser mix (emerald, green Takara)- 10

                               F primer (10pm) - 1µL

                               R. primer (10pm)- 1µL

                                         Template (cDNA )-     2µL

                                           AMQ                        -    5µL 

 

PCR conditions :

 

Initial denaturation - 94˚c- 5mins

           denaturation - 94˚c-1 min

                     anneling- 56˚c--1min

                     extension72˚c- 2 min

                  final extension 74˚c- 15 mins

34 cycles, hold 4˚c- forever



#2 bob1

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Posted 18 March 2014 - 12:29 AM

As random hexamers bind to the RNA randomly, this can interfere with the production of full length cDNA, so you would be better off using either gene specific primers or oligodT for your cDNA production. 



#3 rubhadevi

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Posted 18 March 2014 - 02:28 AM

i was tried by using gene specific primer. but i didn't get amplification. i never used oligodT for to synthesis and i had a doubt whether a problem with gene specific primers. how to check whether primers are good? 



#4 bob1

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Posted 19 March 2014 - 11:34 AM

You can compare the primers to the sequence for your gene found on Genbank.






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