Please Help with Lentiviral Cloning - (May/11/2009 )

I need urgent help with this cloning project because I am getting frustrated and my PI is breathing down my neck because we really need this plasmid to continue.

I am trying to ligate at 2.7kb insert into a 7kb pCDH1-MCS1-EF1-Puro lentiviral vector from Systems bioscience. My 5' ends are not compatible so the first experiements I tried were blunt ending the 5' ends with T4 DNA polymerase (2ul dNTP's <1mM stock>, 0.5ul polymerase at RT for 10min), then isopropanol precipitation, 2nd enzyme digestion for the 3' end (this end is compatible) gel purification and ligation. I tried a 3:1 and 5:1 insert:vector ratios overnight at 14 degrees with T4 DNA ligase. I was using DH5 alpha cells for bacterial transformation and got lots of colonies on my plates (over 100). I tested ~50 colonies and none produced the correct plasmid.

Then I decided to try using PCR to add on the compatible restriction site to my insert. After PCR I cleaned up the PCR reaction, digested with the enzymes and performed a 3:1 insert:vector ratio ligation at RT for 1h with T4 DNA ligase and transformed the plasmid in DH5 alpha cells again with lots of colonies (less than before) and none were the correct plasmid.

Next I got some One-Shot TOP 10 competent cells to try and I used the ligation products from above. I got ~10 colonies/plate and of the 15 colonies that I just tested none were correct.

Does anyone have any other troubleshooting suggestions to make this cloning work? I really need to get my plasmid soon!

Thanks!

I would concentrate on your second approach. Did you add extra bases outside (5') of the RE cut site to your PCR primer? You likely need at least 3; I add 8. Make sure you have a clean PCR product (check length and lack of additional bands on a gel). Column clean up the PCR reaction (I would avoid gels for the cloned DNA). Cut with your REs. If you can, kill the enzymes with heat, otherwise column cleanup the PCR reaction. Your vector should be cut with the same enzymes. If you have a third enzyme that will cut in the middle of the cloning site, you might add that too, to reduce background. Consider SAP treatment of your cut vector, but try it without this also. Heat kill or column cleanup the vector (I would also avoid gels if possible). Mix 1:1 molar ratios at around 30 ng vector in 1 ul T4 ligase buffer + 1 ul ligase + insert + vector + water to 10 ul. Hold at room temperature for 1/2 hour. Transform 1 ul into 50 ul cells. Do a control transformation with 10 pg of pUC19 -- you should get 10-100 colonies.

If you continue to have problems, you should test the ligation efficiency of your insert and vector DNA. Ligate each individually in your ligation mix, heat inactivate the ligase, and run a gel (you'll need to ligate more DNA to see the bands). You should see high MW bands. You can try cutting the ligation with each of your enzymes separately. You should see double length bands. The ratio of single to double length bands in this test shows you the effectiveness of your cuttiing/religation. You should not see ligation, of course, on the SAP treated vector fragments.