I read the table about cleavage close to the end of DNA fragments provided on the NEB website. According to that table efficiency of NotI cleavage of short DNA fragments is 0%. I'm asking 'cause I have problems with my SacI/NotI cloning procedure. Digesting the vector with both enzymes simultaneously is 100% efficient as I'm cutting out an unwanted DNA fragment and purify the linearized vector from the agarose gel. I also simultaneously digest and gel purify my PCR product. After ligation and transformation I get no or just a few (negative) colonies on the selective LB plates. Do you suggest to add further nucleotides at the 5' end of my primer (upstream of the RE site)? Or what might be the problem? I also tried various vector:insert ratios, different ligation temperatures and durations, freshly made competent cells...
Thanks in advance!
https://international.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
Hi, I would suggest you the following:
1) Try to transform other vector or plasmid using your prepared competent cells. Preferaly those plasmid which you have no cut before to see the transformation efficiency of your freshly prepared competent cells. This is to exclude problem lies with your competent cells.
2) Increase plasmid DNA concentration during transformation to maybe 500-1000ng.
Zhi Xiong Chong, thanks for your suggestions. Of course, I tested the competent XL1Blue cells with a standard vector. And they work fine. I only tried to increase the amount of insert, but I will definitely do vice versa!
Dear Sylvia Kohler , we put "GTTGTTGTT" sequence upstream of the RE site in all inserts (both forward and reverse). Maybe it can work for your constructs, too.
Sara Mingu, thanks for your suggestion! I also found publications where they added "CGAT", "GCGC" or "GGCC" in different lengths upstream of the RE site. For me it looks quite random. Is there any rule, specific for every restriction enzyme, which and how many nucleotides need to be added to make the enzyme active? Or do I just have to make sure that the added nucleotides do not interfere with each other or the specific sequence of my primer, respectively?
In addition, in case I have positive colonies, they always show smaller PCR products in a colony PCR than expected (see attached file). When I perform PCR with primers outside of my insert, the products are too small, about 300 bp were lost. When I perform PCR with a gene specific forward primer and a vector primer, I get no results at all or they have the correct size. I'm confused.
Dear Sylvia Kohler , I have not heard of an optimal sequence, unfortunately. In our lab, we only look to make sure that the nucleotides do not interfere with the sequences, as you mentioned. GTT repeats have worked for us so far, however i have heard of other labs around us successfully using random bases as sitting sequence.
In the link you attached there is the following information also: https://www.neb.com/~/media/NebUs/Files/Chart%20image/cleavage_olignucleotides_old.pdf - in this table there are some sequences which have been used with each enzyme. I hope they work for your insert. However i do not know why the researchers chose those specific sequences for each enzyme.
Finally, Gibson assembly may also be useful if other trials also fail.
I prepare my vector as follows: digest with SacI/NotI to cut out an unwanted gene construct, gel purify the vector (with less exposure to UV light as possible), measure DNA concentration. I ran a control agarose gel and digestion is 100% effective. I guess my insert is the problem.
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