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Saadlee's Content

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#180594 Trouble with PCR after cloning

Posted by Saadlee on 01 August 2018 - 02:47 AM in PCR, RT-PCR and Real-Time PCR

I did transformation and found some colonies in the ampicillin plate having Uncut vector and competent cells . And also in the plate with Insert+vector and competent cells. Then, I picked 5 colonies from insert+vector plate and 1 colonies from uncut vector plates. Boiled DNA template was prepared from 6 of them. Then, I did PCR for 6 of them. My positive control was miniprep vector plasmid DNA. The concentration was 300 ng/ul. During PCR, I used 1 ul of template for 7 reaction. I set up 25 ul reaction. Other components were,
10 mM dNTP = 1 ul, taq polymerase = 0.25 ul, 10uM primer (F and R) = 1 ul each, 10x buffer = 5 ul.
The reaction condition was following:
Initial denaturation : 94 for 2 min (1 cycle) , denaturation : 94 for 30 s, Annealing: 55.3 for 30s, elongation: 72 for 45 s ( all three steps for 30 cycles), Final elongation: 72 for 7 min
My expected band size was ~300 bp. There is no band for 6 colonies that I picked from transformed plate. But got two bands for the miniprep vector ( one near 5 kb, other 8 kb but faint). The vector is itself 5 Kb. I think PCR didn't work. I expected to have band around 300 bp. Why those band appears? Is it the problem with designing primer or isolation of plasmid? How to troubleshoot it?

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