Hello
I’ve been trying to clone a gene fragment for some time now, but I'm not getting any results. I did everything according to protocol but I can’t figure out the problem. I amplified my gene then cut both my fragment and plasmid with BamH1/EcoR1 double digest for about 16h (overnight). Restriction sites were designed on the primer. I got strong bands in my gel for both the amplified fragment and the plasmid digest products.
Ligation was done with T4 DNA ligase for 24h with a 5:1 fragment to plasmid ratio. DH5-alpha bacteria was used to make competent cells by CaCl2 treatment and transformation was done with heat shock. Transformation control was positive.
The strange part is that I got almost 60 colonies, which was more than expected. I did colony PCR on 13 colonies at first and got no significant band, so I tried the rest but this time in groups of 5 (5 colonies in each reaction) and still got no result.
I would appreciate any help regarding this problem. Thank you.
did you add 3 extra random bases at the 5' end of the primers? These enzymes often need additional bases for the enzyme to bind to before cutting and without them digestion is very poor
did you add 3 extra random bases at the 5' end of the primers? These enzymes often need additional bases for the enzyme to bind to before cutting and without them digestion is very poor
Are you certain your colony PCR was good and didn't give you false negatives? In other words, did you have a positive control that worked?
Try to purifiy both the PCR product and digested plasmid before T4 ligation.
what are the concentrations of the PCR product and plasmid ??you have to check that very well and try to change the ratios of the ligation in different reactions
also did you digest the PCR product before you purified by PCR clean up kit ??
Thank you all for the responses.
Paul Rutland yes, extra bases were added to the primer.
Michael J. Benedik unfortunately we did not have a positive control. I will check that this time.
Mohamed Halawa we digested the product before clean up.
do you have any plasmid or pcr product that you can use to cut with your 2 enzymes which will show clearly that the enzymes are cutting properly Zahra Fakhraei Ghazvini
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