4 replies to this topic

[yoursisterdebra]

I started doing molecular cloning for the first time about six months ago... it has been something of a crash course but now I *think* I have a reasonably decent grasp on what it is I need to do.  I am attempting to clone a multi restriction enzyme (multi RE) site into the pLKO.3G vector using the vector's EcoRI site.  I ordered the multi RE oligos as phosphorylated 24-mers, annealed them, and checked on a gel to make sure they annealed.  I digest the vector with EcoRI and confirm linearization... I then dephosphorylate with Antarctic Phosphatase.  The AP seems to be working as my "dephosphoylated cut vector + ligase" control transformation gives me only a handful of colonies (less than 10).  My "uncut vector" or "cut NOT dephosphorylated vector + ligase" controls give a lawn of colonies which suggests my competent cells are okay.

My problem is that I can't seem to get my insert into my vector -- every clone I get on my "dephosphorylated cut vector + insert + ligase" plates have been self-ligations of the vector.  At this point I am guessing the problem has to be ligation, since transformation of my controls is working out as expected.  I have tried using different aliquots of ligase buffer and enzyme just in case some of them have "turned" but so far no positive clones.  Since my insert is so small (annealed 24mers) is there any way I can check my ligation product to see if the insert is incorporated?  Could I digest my ligation product with some of the enzymes whose restriction site is located in the insert?

I have tried ligation reactions ranging from 5 microliters to 15 microliters in volume.  I have tried either 50 ng or 100 ng of vector... I have tried a vector:insert molar ratio of 1:1, 1:3, 1:6.  I have tried using 2X ligation buffer and 10X.

I am at the point where the lab manager wants me to do PCR cloning but my boss likes things done the "digest and ligate" way.  I really truly appreciate any help or advice you might have!

Edited by yoursisterdebra, 11 January 2011 - 01:13 PM.

[phage434]

Double check that the annealed fragments have the correct overhangs.  Check the ligase buffer (ATP can go bad).  You should be able to see ligation of the insert with itself on a gel.  Do a ligation with insert only, heat kill the ligase, and run a gel.  You should see multimers from self-ligation.

[perneseblue]

Could you also write down the sequence of the oligoes that you are using? I would like to double check the construction strategy.

[Ameya P]

Could you please elaborate on this?

yoursisterdebra, on 11 January 2011 - 08:01 AM, said:

Since my insert is so small (annealed 24mers) is there any way I can check my ligation product to see if the insert is incorporated?  Could I digest my ligation product with some of the enzymes whose restriction site is located in the insert?

How exactly are you checking if the insert has been ligated into the vector then? I would prefer to do a PCR/ sequencing to confirm the incorporation of the insert, rather than look for some restriction enzyme site.

[perneseblue]

yoursisterdebra, on 11 January 2011 - 08:01 AM, said:

Since my insert is so small (annealed 24mers) is there any way I can check my ligation product to see if the insert is incorporated?  Could I digest my ligation product with some of the enzymes whose restriction site is located in the insert?

Yes you can. Just make sure that the empty plasmid is not cut by the enzyme that you use. So if the enzyme cuts the insert is present. If it does not cut the insert is absent.

You might also want to try a double digest with a second enzymes that cuts some distance away from the insert.  In this instance, a single band would indicate the insert is absent. 2 DNA fragment would indicate the insert is present.