I use this kit trying to clone a gene 1011 bp in size into a shuttle vector 6000 bp. I got transformants on the plates but after the screening using either sequencing or colony PCR nothing there is related to my gene of interest? I am just looking for any advice about troubleshooting as i used all the points mentioned in the company handout nothing helped. I repeated the whole process three time now.
Thanks
Hi, So sorry for your bitter experience with in-fusion cloning experiments. I want to make 2 suggestion:
1. You need to make sure that your primers are well designed to contain 15 nt base homology with the linearised vector. Follow the universal primer design guide for in fusion cloning meticulously.
2. Your method of linearising your vector is very important. It is always recommended to use double digestion with restriction enzymes that produce like 4 nt overhangs. With that, the chance of getting positive clones is increased.
Finally, you should always have a negative control to make sure your procedure is correct.
Cheers
Hi, I also have trouble with in-fusion quite often. Other than the points made above by Muhammad, which are all correct, I found that results vary depending how you screen for positive clones. I have tried different methods for PCR detection of positive colonies and have found that the polymerase mix you use is very important. I grow colonies in 150microL of media with selection for about 3-4 hours then use 1microL of culture in the PCR reaction and use either Phire Mastermix or Taq polymerase and T7 promoter primer and gene-specific reverse primer. Results vary depending on the insert. Sometimes the T7 primer is not good and will give me no positive colonies but I try again with gene-specific forward and reverse and then I get positive results.
Thank you Mohammed and Anna for your useful advice.
I do always have negative controls and it looks like what it should be very few colonies compared to the transformation plates where i have hundreds of them. I will try the way of screening you mentioned and hopefully It gonna work.
What would you do if there is no colonies at all?:( Transformation efficiency of competent cells seems very good.
Hi, Finally it worked and it seems that the way I screen the transformants is the problem. This time I compared the size of the few colonies on the negative control plate to the size of those on the transformants plates and picked the different size confirmed it by double and single digestion and i got my gene inserted into the plasmid!!
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