There is a band of same size as that of my insert in the negative control PCR reaction,though it is much less intense than the other colonies. What could be the explanation?
Best case scenario, you had a spillover from a neighboring well when loading your gel. To rule this out, load your negative control first & leave a few empty wells between the negative and the next sample.
Most likely scenario, you have contamination. Toss ALL of your reagents (buffer, enzyme, water, TE, primers) and remake from new stocks.
Katie A Burnette and Abhijeet Singh are correct contamination is the most likely explanation. I would agree that you should toss all of your reagents. When working with plasmids the pipettes can also be contaminated. You should clean them regularly and using filter tips is ideal. I would agree with replacing all of your reagents. If you want you can setup an experiment to try to find the source of contamination basically setting up a PCR with all new reagents except for one of the old reagents for each sample. This can be useful if you don't want to potentially throw out expensive reagents such as enzyme. If the pipettes are the source of contamination then you will have to toss all reagents likely and send the pipettes off to be cleaned.
Hej Katie and Joshua,
She is just a master student and likely not very experienced in lab.
I guess its just the cross sample contamination. So let her do the PCR again with care before letting her throw away all the reagents. I could be an expensive advise to simply throw away reagents without any idea of what is the actual reason.
Its possibly due to contamination during setting up the reaction or may be the reagents are contaminated... I wud suggest you to re-pcr and if possible use new reagents to check if the reagents are contaminated or not.
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