Simple sublconing not working - why? (Oct/24/2007 )

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So I have this simple subcloning that isn't working. I consider myself usually fairly competent in doing these types of things...

I PCR up my gene of interest using PCR primers that contain restriction sites (Xho1, Xma1) and everything looks good when I gel extracted it.

I then digested my vector and PCR products with these two enzymes, gel extracted (everything looked ok), and tried to ligate them together. I just got background (either uncut vector or uncut/religated) as the numbers of colonies (about 10-15 in all plates) were the same in the control, no insert (cut vector only) ligation reaction. I also verified this by minipreps.

I checked to make sure my enzymes cut the vector by a linearization assay of the plasmid and it looked like XmaI cut slowly (but cut ok). Based on this I redigested my insert and plasmid and tried cloning again. This time I got very few colonies (like 2-3) in all plates. these were also just background.

So I verify at nearly every step by looking at the gel before extraction to see if things are working and it appears they are. The cells are competent, the enzymes cut the vector, the ligase works. Huh..

the only thing is that I have no way of verifying if my enzymes just can't cut the inserts. I left 3 bps at the ends before the restriction sites on my primers which should be sufficient acccording to NEB (they say 1-2 works with 97% efficiency) but I worry nonetheless. Should I try new PCR primers with a more bps before the RE sites?

What do you think?
Any advice appreciated,
Mountainman

-Mountainman-

Two things to check. First, you may be trashing your DNA with UV during the gel extraction. You might want to try ligating without gel purifying. Second, how are you purifying after the PCR? PCR enzymes and dNTPs can fill in cut ends unless they are completely removed prior to cutting with restriciton enzymes.

-phage434-

QUOTE (Mountainman @ Oct 25 2007, 12:45 AM)

First subclone the PCR product without digestion into systems like Zero blunt (Invitrogen). This works very efficiently. From this vector you can generate larger quantities of the insert and verify the restriction digest also on a gel. And additionally cloning of cut inserts from vectors is much more effitient than cloning PCR products.

-Dukes-

QUOTE (phage434 @ Oct 24 2007, 11:15 PM)

I suppose I could try purifying the PCR directly on a Qiagen column since it seems specific. I gel purify my PCR products with a Qiagen gel extraction kit after the PCR but before I cut them, which should remove the enzymes and dNTPs. Then I gel purify again after I cut the PCR products.

I thought a UV transilluminator used a low intensity uV (it is definitely less bright) so you could use the bands to clone. It can nuke my DNA?

Do you think 3bp is enough...I'm worried I may have to make new primers with more bps before the cut ends.

Thanks,
Mountainman

-Mountainman-

I find sometimes that it proves best to revert to the basics:

1. Test the efficiency of your selective antibiotic. Your negative control shouldn't grow at all.

2. How effective is your transformation...eg, what type of cells are you using?

3. Is your DNA pure enough? If you have access to an ultra, try cesium chloride DNA preps.

4. (I've had trouble with this one) CHECK your LIGASE! They're notorious for degrading.

5. How effective is your polymerase? Your dNTP's?

Also, if you're using NEB restriction enzymes, you can surely take their word for their efficacy. Top notch, they are.

-gunnerpiaffe-

selctive antibiotic works...transformation works (i did uncut plasmid and nearly got a lawn)...Qiagen columns have always given me pure enough DNA in the past....PCR works......

I suppose I could have a bad lot of ligase though....that would be an attractive explanation since I it would be the leas work..

UPDATE: I am transforming new ligation with fresh ligase now.

-Mountainman-

Sounds like something simple. Check your primer sequences, make sure you haven't stuffed up the RE site design.

Here's a couple of more obscure suggestions:
-incorrect vector - digest the uncut vector in 3 or 4 places to get a couple of confirming bands on a gel
-incorrect insert - digest the insert in the middle to get confirming half-sized bands

I'd be interested to know the solution once you find it.

Seeya, Rob

-killerkoz17-

QUOTE (Mountainman @ Oct 25 2007, 04:21 PM)

UPDATE: I am transforming new ligation with fresh ligase now.

Hi, a couple of ideas.
As is been suggested I will clone PCR on a blunt vector, much easier to digest and purify your insert. If this is not appealing to you (extra cloning steps), I will suggest that after digesting your PCR product (which you have gel purified) don't gel purify again. You can use QIAgen gel extraction kit to remove restriction enzymes without having to run the gel, I think you might be loosing to much PCR product by gel-purifying twice. Have you tried different insert-vector ratios? Do you dephosphorylate your vector?

Hope this helps, good luck!

let us know when it works!

-almost a doctor-

So I tried a new ligase but I can't determine whether that is the problem or not because the competent cells I got from a colleague don't work even with uncut plasmid. I ran out of competent cells of my own...

I think I will give her some DNA to try to transform to see if it is a protocol thing or something but I think her cells are dead.
I will make my own cells and try again with my frozen ligations and if that doesn't work I'm going to use a PCR cloning vector (with U or T overhangs). This is ridiculous! That's what I get for jumping into cloning in a new lab. I guess it takes some time to get settled.

To answer your questions. I sue XmaI, XhoI so I shouldn't need to dePhosphorylate. I seem to still have enough DNA after all the purifications. Vector is at 17ng/ul and inserts range from 17-29ng/ul. Last ligation I used 17ng of vector with either 54ng (3:1) or 85ng (5:1) insert

Question...how much easier is it to use an extra step with a PCR cloning vector. The ligation is more efficient somehow?

-Mountainman-

I got the similar trouble.

I did the same process to subclone my insert to a expression vector. I did several subclone before with the same process. but it won't work in this new lab. I do not know where is the point. I add 3 extra bp to the outside of the enzyme site for digestion according NEB website suggestion (it works for my previous work). But I cannot get the result. I suspect many things but I cannot change all the staff for the single construct as I am the new guy here although no one else works on this cloning staff now.

I called NEB. They suggestion add 6 bp for sufficent digestion. Anyone have suggestion for the 6bp. what should I take? GCTACG?

After transformaion, I cannot get any colonies on the plate. So I just worry about my ligase and competent cells instead of my digestion.

Question, can I use qiagen gel extract kit or PCR extract kit to remove digestion staff and enzyme instead of run a gel? which kit works better?

-newboy-

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