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Gam8Doc 05-11-2009 02:52 PM

Trouble with Cloning into Lentiviral Vector
I am trying to ligate my insert into the pCDH1-MCS1-EF1-Puro vector from System Biosciences. My insert is 2.7kb and the vector is 7kb. At first I was trying to do a blunt end ligation on the 5' end and a cohesive (sticky end) ligation on the 3' end and tried a insert:vector ratio of 3:1 and 5:1 (Overnight ligation at 14 degrees with T4 DNA ligase) with no success. I then used PCR to add on the complimentary cohesive site to the 5' end of my insert and amplify the fragment and ligate these two pieces together (1h ligation at RT with T4 DNA ligase). At first I was using DH5 alpha competent cells and just recently got some One Shot TOP 10 competent cells. With the DH5 cells I would get lots of colonies/plate (over 100) and the colonies that I tested (~50) did not produce the correct plasmid and ~10 colonies/plate with the TOP 10 cells and again none produced the plasmid.

Can anyone help trouble shoot this with me. I am getting very frustrated and my PI is breathing down my neck because we really need this plasmid to continue our work.

Also has anyone used the pLenti-promoter plasmid from Applied Biological Materials? We are thinking of trying this plasmid next.

Thanks for any suggestions!

millartime 07-07-2009 02:12 PM

Re: Trouble with Cloning into Lentiviral Vector
I have also recently been trying to clone genes into lentiviral vectors with limited success. I have been using the pLVX plasmid from Clontech and also an in house vector from another group doing lentiviral work. At first, the restriction digestions worked perfectly and although we had to use a UV box, we cut the vector and insert from the gel an tried ligating into competent cells from Bioline (efficiency 10^7). This gave us no colonies and after changing everything that we used for digestion and ligation, I tried to use BL21 that we made in house. This gave us many colonies that had a vector but no inserted gene of interest.

A colleague from another lab suggested that she try to do the same work using her enzymes, lab equipment etc. The only thing she used differently from us was the bacteria she transformed. They are Stbl3 competent cells from Invitrogen. These are specifically made for transforming awkward vectors such as the lentiviral vectors. After she had done the work, it was successful (much to my annoyance although I was slightly relieved as well!) and we are currently trying to make the virus with the new vector she has created.

You may want to try using the Stbl3 cells fro your transformation although I haven't had a chance to test them myself. Our problems appear to be quite similar and as far as I can see, the only difference between the way we are doing things (not working) and how my colleague is doing things (working) is the bacteria.

Davis Thangjam 02-16-2013 08:58 AM

Re: Trouble with Cloning into Lentiviral Vector
I am facing a big problem with cloning. My vector is pLVX-puro(8kb) and my insert size(1kb).....after transformation no colonies were seen......if few colonies were screened the plasmid vector is wrong......I do get positive results with my vector..the efficiency is high (10*7)......
Vector (50ng)
Insert (150ng)

I keep on repeating but cant get the result....I even change enzyme(NEB),comp cells (IMTEC DH5alpha and Agilent XL1 comp cells),Ligase (thermo and Roche) but no effective result.

Please suggest something.....:(

Sofia76 06-11-2013 02:17 PM

Re: Trouble with Cloning into Lentiviral Vector
I'm having problems with cloning too. I'm using pLVX-puro vector from Clontech (I guess the same as Davis) which is 8102 bp and my insert is 1675 bp. We use FastDigest enzymes and T4 ligase from ThermoScientific. Competent cells are JM109 from Promega. We got a lot of colonies but vector is empty. Anyone has any ideas? Davis, did you succeed? Thanks

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