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[ram]

I am working with expression cloning of a gene whose size is ~1.7kb. For expression cloning I used pET vector. I double digested plasmid as well as insert with EcoRI and NotI REs and gel purified both. This purified plasmid and insert have been used for ligation in 1:1 molar ratio. After transformation i got many colonies, but when i performed colony PCR to confirm the insert i got very high non specific amplification, which i have never ever experienced before for any colony PCR. for colony PCR i used vector specific primers. Following pic shows the colony pcr with six clones having non specific amplification



With this result i tried cloning the gene in TOPO TA cloning vector (invitrogen). I did PCR to get amplicon of my interest which was A tailed and used for cloning. Ligation reaction was kept at RT for 30 min. reaction additions was performed as per the manual. After transformation I got many colonies. I have chosen few of them and did colony PCR with vector specific primers. Here also I got very high non-specific amplification. Following pic gives the clear idea about the PCR.



To check whether there is any problem with colony PCR cocktail (contamination!!), i did one trial PCR using same cocktail with cloned TOPO TA vector from my colleague. Also I put one blank reaction (without template). This time it didn't showed any non-specific amplification also there was no band in blank. this means that the PCR cocktail is OK and working fine. Following is the pic to explain!


with this background, i am unable to get the reason behind such non specific amplification. why i am not getting cloned fragment in both the vectors?
Do this happen while you go for expression cloning? OR there is some problem with vector system?

[gyma]

for colony PCR, you should use the primer set in which one is specific for vector sequence while the other is for the insert.
in my experience, I usually get a lot of false positive results even using the primer set I mentioned above. so I dont do colony PCR ever since. Now I just pick all the white colonies (b/w selection), extract plasmid and check the size on the gel. no false positive anymore.