Dear All
I am working on Baculovirus expression system. I cloned the gene of interest in pFASTBACK. Then I transformed this plasmid in DH10Bac bacteria to make the bacmid.
I extracted the bacmids from white and blue colonies and did the PCR using either M13 Forward and Reverse primers or M13 Forward and gene specific Reverse primers (attached). White colony shows the fragment with the right size with both pairs of primers. However, in blue colonies I expected to see 300 bp fragment but there is no band. I also expected to have no band using M13 forward and gene specific reverse primer. This pattern exists in different blue colonies.
Based on the bands here, the primers bind to the template correctly (I think). So, what could be a reason for this in blue colonies?
Does anyone have any idea of what is going on here?
Any comments would be much appreciated.
The white colonies are formed by single cell correctly transposioned in the cell containing both wt bacmid and the fasbac plasmid. In the right selection plate, the blue colonies host both the wt bacmid and the plasmid. As the transposition constantly happens in the cells inside the blue colony, you would never get a pure wt bacmid in the blue colony without transposition in some cells, unless you do not use pFastbac plasmid and the amtibiotics. People sometimes to use blue colony to streakout into new selection plate to get white colonies in case the transposition failed to get white.
Thanks for the comment.
So do you think it is a good idea to go to the next step?
we did such confirm many years ago, never got negative results. Now, we ignore this confirmation, and so far, we never get into problem without confirmation by PCR.
So if there are some transpositions why there is no band when M13 primers are used for PCR? while M13 forward and gene specific reverse shows a band.
try to test this PCR with your wt bacmid. likely it's due to the PCR itself if you also cannot make it work with your wt bacmid. PCR in many cases, represents "problem comes regularly".
I also agree with Mr. Zhang. You can check the blue colonies in a new plate which contains all necessary antibiotics. And you can additionally use M13 and Gentamicin Reverse primers.
Hi Amit, can u plz share me ur pcr reaction condition. I am getting multiple band in my bacmid PCr. I tried few condition but did not work. I am not sure whether purification of my bacmid is good or not. I got 2000-5000 ng/ul DNA for bacmid after purification. How do u check if there is contamination with RNA?
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