Random (and frequent) transposon insertion
Hello everyone,
This is my first post here, but I read the SEQanswers forum since a while and I can already say thank you for all the good hints I found here.
I am trying to randomly insert short pieces of DNA (200-300 bp) into a target gDNA (7 x 10e7 bp long). Ideally, I want to have one insertion every 5 - 10 kb.
I (very naively) first thought of using a Tn5 transposase complexed with a single transposon (e.g. EZ-Tn, Lucigen). Therefore, I purified the Tn5 as in Picelli et al 2014 and I complexed it with my custom transposons. However, transposition is not that frequent. I know that my protein is active because when complexed with Nextera adapters (for a tagmentation reaction) it works beautifully.
Do you know how I can increase the frequency of Tn5 transposition?
If not, can you suggest any alternative tools (other transposon system maybe)?
Thanks.
—
Claudia
This is my first post here, but I read the SEQanswers forum since a while and I can already say thank you for all the good hints I found here.
I am trying to randomly insert short pieces of DNA (200-300 bp) into a target gDNA (7 x 10e7 bp long). Ideally, I want to have one insertion every 5 - 10 kb.
I (very naively) first thought of using a Tn5 transposase complexed with a single transposon (e.g. EZ-Tn, Lucigen). Therefore, I purified the Tn5 as in Picelli et al 2014 and I complexed it with my custom transposons. However, transposition is not that frequent. I know that my protein is active because when complexed with Nextera adapters (for a tagmentation reaction) it works beautifully.
Do you know how I can increase the frequency of Tn5 transposition?
If not, can you suggest any alternative tools (other transposon system maybe)?
Thanks.
—
Claudia