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expression cloning in TOPO TA and pET vectors

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#1 ram



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Posted 25 April 2010 - 07:26 AM

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

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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.

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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!
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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?
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#2 gyma



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Posted 25 April 2010 - 11:43 AM

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.

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