I designed some primers to amplify a tomato gene. When checked two primer pairs worked very well and gave me required length bands. I repeated this experiment three times and always get bands of same length. Now when i want to clone that gene i set a PCR again but did not get any band. Its really surprising?After getting failure many times I changed many things e.g. new primer stock, new taq, dNTP, fresh extracted tomato genomic DNA, changed the water.Even i used ready master mix but did not get any band except primer dimers( some bandsof almost 100bp where as my gene is 1400 bp) I could not figure out the problem. I need suggestions and help to resolve this problem.
Thanks in advance.
Do you try repeat the step clone? Or confirm that your clone is ok?
I did not clone yet. I want to amplify the gene fresh then use for cloning but i am not getting the amplified band which i got before.
Do you try in other temperatures, because you said that use a new primer stock?
Yes i tried the range of temp from 59C to 64C. Initially i got result at 63C.
Maybe this temp is very high and denaturing a tomato gene. The first time maybe was luck. In this moment i don't see other problem...
Thanks Thiago Onofre for your replies, I will try with reduced temp.
@Thiago Onofre i got my desired bands. Its surprising ; My lab has 5 thermocyclers i when i am using other i am not getting any band but one of them is giving me desired bands.All thermocyclers are in good condition and other people use them.
strange... it's very strange, but good. That is good this your thermocyclers give you desired bands. Good job.
Do you have any idea that why this is happening? I tested it two times today., Reaction mix was prepared in one tube and after distributing in other tubes i used different thermocyclers with same programme.
Are the thermocyclers same mark? Because it is false, exist the possibilit of diferences between temp (diferent firm = diferente calibrate) and the tomato gene can be very sensible. What do you think?
Yes it is possible that different brands may have different calibrations; We have 3 BioRad I cyclers, eppendorff's Gradient and ABi Biosynthesis. Interestingly i am getting bands by using one of BioRad thermocycler. Perhaps it has some problem internally and the temp it is showing on screen(and feeded in programme) is not exactly as in the wells of thermocycler block. Thats why i am getting the bands.
Hello Doctor Muhandas,
Did you find what is going on? I have the same problem. Did you have any good results doing any modification on the protocol?
Thanks
H.J.
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