At first when I started colony cloning a 270 BP insert into 5677 BP vector( pet sumo adapt) I had a lot of problems in confirming my positive clone using colony pcr.
I did colony pcr with t7 promoter and terminator but all colonies gave the same size band as that of the vector . So I assumed all of them are empty vectors still i was a bit sceptical because i had tried it a number of times and with everything possible I could with all the control .
today I just tried a colony pcr with the gene specific primers ( the one that I Initially used to amplity the insert). lane one is just digestion of the plasmid prep from ligation plate with bamh1 and bsa1.( don’t see any 270bp band)
whereas the last 3 wells show me a band at 270 when pcr with gene specific primer .
there is also some band just below the well , and I don’t know what it is, is it the genomic dna amplification ?
how is it possible then t7 failed to show me a positive clone in the sense that vector and assumed insert had the same size amplification on gel but somehow I see a band with gene specific primer . How is it possible .? Also what could be that extra band right below the gel comb
The upper bands look like genomic DNA. How much DNA from the colony had you added to PCR?
And regarding primers, I would suggest that your primers are in wrong direction, so instead of amplifying from promoter to terminator (resulting insertion) they work from terminator to promoter (resulting empty vector). If you designed them yourself, try to use their reverse complements. If they are commercial, check documentation and make sure that you understand their purpose right.
Yes, the upper bands should come from bacterial nucleic acid. This is quite common when doing colony PCR.
About the PCR with primers annealing to the vector, what is the distance between the two annealing sites in the empty vector? Are you sure that this length is theoretically distinguishable from the one in recombinant plasmids?
Did you include a negative control (empty vector) in yous PCR with specific primers to rule out any non-desired amplification products on the vector backbone?
As already mentioned, don't worry about the large band. Just prepare a miniprep of one of your positive clones and get the sequence of your insert. In case it is positive, everything is fine, in case it is negative your specific amplification was a result of a contamination. In that case you should re-check your strategy.
Perhaps there are fortuite annealing sites for your specific primers in the vector. Probably match is nor perfect. I remember that the specific bands were weak. Thats why this control is relevant when using specific primers. . The other gel you showed with vector primers indicates that clones are not recombinant. Anyway, if you are not yet sure, sequencing can give you the final answer.
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