I want to clone my 60 bp insert into my approximately 7000 bp vector. I did the ligation with a vector to insert ratio of 1:10. Either very few or no colonies grew. Also my colony PCR results were negative. How should I change the vector insert ratio?
If your vector is dephosphorylated, ensure that the 5' end of your insert is phosphorylated. It is common for synthetic DNA or PCR products to lack phosphorylated 5' ends.
You could try other insert/vector ratios. I have previously used ratios of 1:3 and 1:5.
Is your vector commercial or prepared by you?
Is the band a PCR product, a synthetic hybridization product, or extracted from another plasmid?
Is your ligation reaction blunt or sticky?
What is the compatibility of the DNA ends?
All this information is important for providing a more accurate response.
Hello Elif, I agree with Eddy— we need more information to provide useful feedback. Sounds like your competent cells or protocol for transformation may be part of the issue. Have others in your lab had issues with those cells? Could you attach the protocol you used? Honestly, your insert seems small enough that you may just be able to order two primers to do your cloning. For example, each primer could be 45 nucleotides long (just making a number up) where 15 anneal to the vector and the other 30 are part of your insert. Following PCR, just do a DpnI digestion, PCR cleanup, ligation, and then transform your cells. You would need to account for melting temperatures, secondary structures, etc. given the length of the primers, but just an idea. You could also try Rec-A independent recombination or RAIR cloning, see the publication below. Most common laboratory strains of E. coli are capable of RAIR. All you would need to do is buy four primers, two to amplify the vector and two to amplify the insert, with the 5’ and 3’ ends being homologous to one another. After PCR you just add 1-2 microliters of each reaction to heat shock cells, carry out heat shock protocol, plate the cells, and do colony PCR the next day to screen for successful clones. Good luck!
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Hello! So for really small stuff it is better to use QuickChange mutagenesis. This procedure works really well. It was a product from Stratagene, but I think they went bankrupt. In the manual, it is the "primer" design (which is your 60nt insert) that is important. You can get pretty much all of the enzymes from NEB or any other general supplier. High quality competent cells, like Top10 will also help. Please have a look at this manual.
Eddy Ernesto Gonzalez Horta Jamison Drew Law
Hello, first of all thanks for your feedback. I bought my vector commercially. The 3‘ and 5’ ends of my insert are designed according to the restriction sites in my vector and they make a sticky end. We also use the heat shock protocol in transformation. Also, we have never had such a problem in transformation as a team before, but we have never used such a short insert.
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