Can't seem to get insert into vector... please help - (Dec/02/2010 )

I am trying to insert a DNA oligo encoding a shRNA into a vector (pLKO.3G -- http://www.addgene.org/pgvec1?f=c&cmd=findpl&identifier=14748 ). Another postdoc in the lab had used this vector previously to insert an shRNA sequence using the same scheme I am following and had it work. However, he is also not the most organized individual... at one point he gave me DNA for the wrong vector which I realized when I checked my digestions on gel (thank goodness!). For insert, I am ordering DNA oligos as sense and antisense oligos and then annealing them (heating in a beaker of boiling water for 4 minutes, allow to slowly cool to room temperature). I have tried two different methods that as far as I understand things should work:

1. Digest pLKO.3G vector with PacI and Eco RI (sequentially) to produce non-compatible sticky ends. I know the vector is linearizing with each enzyme (1st cut) by running a gel but I cannot confirm the release of the intervening sequence (2nd cut) as it is very short (23 bases). However, if I cut with PacI and then run a gel the vector is linearized; likewise, if I cut with EcoRI and then run a gel the vector is also linearized. So I feel pretty comfortable that the sequential digestion should leave me with my desired ends. I am then ligating my vector and insert (overnight ligation) and transforming. I only get re-ligated vector each time. We use the C2988 competent cells from NEB and I follow the transformation protocol provided with them.

2. After repeated attempts using method #1 and only getting self-ligated vector (no insert), my boss decided he thinks PacI is the problem (no real reason other than he is not a PacI fan). So, we designed a multi-restriction enzyme (multiRE) insert to incorporate into the vector when it is singly cut by EcoRI. This multiRE insert can then be used for cutting and ligating in our shRNA inserts. I designed the two oligos for the insert to have EcoRI overhangs when annealed, ordered them phosphorylated (since I would need to dephosphorylate my vector to prevent re-ligation), and annealed them. I cut my vector with EcoRI, dephosphorylated with SAP, and performed a ligation reaction with my insert. Still just self-ligated vector. We were concerned about the SAP as our vial was pretty old so I used newly-ordered SAP and tried a variety of incubation times (10 minutes to 1.5 hours!) but still just self-ligations upon transformation.

We THEN thought the phosphorylation of the oligos might be an issue as lately we have had a number of problems with our oligo-producing service. I thus designed a longer version of my original oligo which incorporated ApoI sites towards the end as well as additional non-important bases to allow the ApoI enzyme room to work. My goal was to anneal the oligos and then digest with ApoI -- thus producing sticky ends that would be compatible with the EcoRI overhangs of my vector AND would be phosphorylated. However, yet again I can't seem to get my insert into my vector and transformation yields only re-ligated vector. Ligation reactions of dephosphorylated vector (with ligase but no insert) yield maybe 10-15 colonies. My vector + insert transformations only yield maybe 20-25 colonies. In method 1, I was getting many hundreds of colonies from my vector + insert plates.

I am kind-of at my wits' end at this point... I am relatively new to cloning (only a few months) and while this has been valuable for teaching me some of the basic concepts I would really like to start my shRNA knockdowns! And I don't want my new boss to think I am a moron for not being able to do a simple cloning experiment. I would sincerely appreciate any advice or ideas you might have.

Thank you very much, and thank you so much for participating in this forum. As a newbie to molecular cloning this site has been a tremendous resource!

How old is your t4 ligase buffer? If you have freeze-thawed it more than twice, you will need to add fresh ATP (or aliquot it the first time you thaw).

I have found it is best to clean up the digests after each digestion, otherwise it seems that there is some steric hindrance from the enzymes still being stuck on the DNA when you go to do the second digest.

You can try an alternative way to produce the cut vector. Make primers to the vector, aimed outward from the desired cloning site, and including the cut sites and 5' overhang needed to allow digestion. Amplify to produce a linear PCR product. Digest with DpnI, which will cut the plasmid you used as a template for the PCR, but leave the PCR product unchanged. Purify the PCR product (Important!) and then cut with your enzymes. The background plasmid should be very low with this approach, and it should be easy to avoid the religated plasmids you are seeing.

bob1 on Fri Dec 3 00:34:21 2010 said:

Hi, that is a very good point about the T4 ligase buffer. This last ligation was done with a totally new tube (never thawed before) so that should not have been a problem. However, I will take your advice and aliquot from here on out!

I forgot to mention it but I do column-purify my digests after each enzymatic reaction.

yoursisterdebra on Fri Dec 3 12:31:46 2010 said:

Column purification is inadequate -- it will not remove uncut vector, small digestion fragments, etc. You need to recover just the linearized vector and the digested insert -- use gel purification to recover your pieces.