Here is my dilemma:
I am trying to insert a 3.7kb sequence into the plasmid pMA3211 (for tet-inducible expression of insert sequence), which is 6.7kb. First, I digested pMA3211with SmaI (blunt) and then double-digested the donor plasmid with Afl II (overhang) and EcoRV (blunt) for 2h. After deactivating the restriction enzymes and letting mixtures cool down, I added Klenow polymerase (with dNTPs) to the donor plasmid mix and antarctic phosphatase to the pMA3211 digestion. After the next deactivation step, I gel-purified both the digested insert and vector and set up o/n ligation at 16C. For the ligation step (total volume 20ul), I've been trying between 15-60ng of vector with 1:3, 1:5, or 1:10 ratio vector/insert. My transformation protocol is to heat shock 5ul of ligation mix into 25ul of NEB-5-alpha cells (allow mix on ice for 15 minutes, heat shock at 42C for 30sec, 5 min recovery on ice) and then plate cells after 1h recovery.
My results so far: either I get no transformants on selective media plates (ampicillin) or I get a handful of transformants in approximately equal numbers on both vector and insert ligation plates. After miniprepping the colonies from the insert plate and performing digestion analysis, there's no evidence of an insert.
Any optimization suggestions or information about places where I could be fowling up the cloning would be useful. Could there be a nuance about inserting such a large insert into a vector only twice the insert's size that I'm missing?
Thank you.
Hi,
Briefly: Blunt end ligation is quiet tricky. Compared to cohesive ligation the efficiency very much lower. and since the size of insert is more then 3 kB. Your ligation is on the far side of complexity. And you also transform with 5µL of ligation mix in 25 µL of competent cells (this 1:5). this might also be very stressful (shock) for the competent cells. You also do a couple of pre-treatments. Some suggestions are:
-start with a massive amount of products
-I would skip phosphatase treatment (since you gel-purify, so you get rid of WT plasmids)
-ensure blunting is done well
-I would use T4 ligation at RT (see manufacture manual e.g. NEB) for at least 2 hrs
-I would use 5 µL of Ligation mix to transform 100 µL competent cells
-Don't forget controls though.
-Gel-purify using TAE (NOT TBE, keep in mind that some acid might inhibit T4 ligase activity)
-RE-digest with at least two different REs
Cheers and goodluck
TS.
This seems to be the answer of the day today, but...
Have you thought about using a Gibson assembly? It's a very, very useful procedure for dealing with tricky ligation reactions.
All the above are very useful hints, but for me there are two critical points:
1-¿Which is the efficiency of your competent cells? I would advise to use the highest commercially available or even electrocompetent cells (for electroporation). Using 10e9 efficiency instead of 10e8 means 10-fold more recombinants!!
2.- As Terrens said blunt ligations are dammned difficult. I would reduce the ligation volume to 10 ul (or even to 5 ul :1 ul plasmid+1 ul insert+ 0.5 ul 10x buffer+ 2.5 ul H2O), ligate o/n at 12 ºC and transform as Terrens suggested. Also, as Andrei said, you can use PEG4000, or spermidine to 1 mM (final concentration). These are used as "crowding agents" to reduce the effective ligation volume. Be advised that ligation requires ATP, so for a difficult ligation use a fresh lot of ligation buffer.
Good luck!!
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