TA Vector to pQE Vector chang problem - Can't get rid of originally TA vector.. (Aug/02/2005 )
I 've done the TA cloning(PCR product ligate to yT&A vector), and I am trying to cut my insert from TA vector and ligate it to pQE-30 expression vector(QIAGEN) . Everything seems well because I use KpnI and SalI to cut my originally TA vector and gain the right sized inserts, and I also cut new vector(pQE vector) at the same time. After restrictin enzyme digestion, I do gel purification, and pick 2 ul to check purified DNA is still there and is single band. Then I do ligation and transformation and clony PCR to screen transformants and collect plasmids.
Then strange thing happened!! Because after I check my newly plasmids, most of them are old vector size(3.9kb=TA vector(2.7kb)+inserts(1.2kb) ), and very few of them are predicted size(4.6kb=pQE vector(3.4kb)+insert(1.2kb)), and I sequece those vectors(include 4.6kb (pQE size) vector) then found them are still TA vectors!!!! Then I do the experiment again(from the restriction cutting) still gain the same results!!
I am stuck with vector change problem for nearly 1 month, please give me advices what should I do...because I don't know how to improve... thank you!!
It sounds like you have some uncut vector in your gel purified fragments. I would advice gel isolating your insert again (ie do 2x gel purifications).
Improved automated DNA sequencing