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ROHIT

Member

Registered: 2025-02-09

Posts: 1

SECONDARY REACTION NOT WORKING

MY insert is 876 bp.I am trying to amplify gene from bacteria genome and insert it to plasmid having 5851 bp.My forward and reverse primers are ACCAACAAGGACCATAGCATATATGTTCACGGGAAGTATTGTCG & GTGATGAGATCGAAGATCTGAACCCAGCAAACCGGCATGCTTA respectively.For primary reaction i am directly picking colony from plate as source for bacterial genome.Primary PCR coditions are
94 for 5 min
98 for 1 min
98 for 30 sec
59 for 1 min
72 for 1 min 30 sec
30 cycles
72 for 5 min.
I am getting almost exact bp as suppose to get in agarose gel.One problem is after pcr purification conc is less around 11 ng.So i did amplyfy again with primers,now my conc is good to proceed for secondary reaction.For second reaction
i using this condtions below
98 for 30 sec
98 for 30 sec
57 for 1 min
72 for 5 min
But in gel it is smeared band.Please help me fixing this.Thank you.