hello all
I hope someone can help
I have been trying to subclone my 2kb insert from a vector cut with BamH I/Pme I into pVL 1392 9.6 kb cut with Bgl II/Sma I
this is a sticky / blunt end clonig
I have been trying 1:5, 1:10 vector : insert ratio
I have been using takara mighty mix reagent ligase mix
each time I got clones, check the miniprep by restricition enzyme digestion I found 3.9 kb insert instead of my insert
can anyone help regarding this problem
I have changed all the reagents , use another d.d H2O , plenty of RE
increase the DNA concentration
every time I check for the vector insert complete digestion , gel purify and ethanol precipitate my DNA and reconstitute in TE buffer
I also tried yeast tRNA as a carrier to increase the transformation efficiency ,..but in vain
I hope you can help me how to get my insert into this vector
thank you a lot

What enzymes do you use to verify miniprep? remember you have modified the sites and the ones initially used for digestion are no longer present.