cloning problems - help! (Oct/18/2005 )

Okay, down to business...

I've been attempting a cloning operation for a couple weeks now. I'm cloning 4 PCR products (from here know as M1, M2, M3 and M4). Their sizes are as follows:

M1 ~ 980bp
M2 ~ 650bp
M3 ~ 250bp
M4 ~ 150bp

Now, to put myself through the entire process at least once to get my head around it, I did my entire work through with M1 from start to finish:

PCR amplification (I have primers for M!, M2, M3 and M4 already)
Gel check PCR products (lots there... combined 5 tubes)
Gel purified PCR product and ran a small aliquot on a gel to ensure it's still there
double-digested PCR product (with NdeI/SacI) as well as my vector, pHTT7K
performed my ligation reaction
electroporated electrocompetent DH5-alpha cells and plated them (using insert:vector ratios of 3:1, 4:1 and 5:1)
plated bacteria on kanamycin plates (as pHTT7K contains kanamycin resistance)
mini digested 30 broth cultures grown up from 30 single colonies from these plates (lots of colonies)
20% of the colonies contained the M1 insert (6/30)

Hurray for success!

Now here's where it gets weird!

Now that I ran through all this once, I decided to do the rest together rather than one at a time. I followed the exact same procedures that I used in the layout for M1, but I'm not getting ANY positive colonies with insert in them! I've tried 3 times now and it's driving me bonkers. Here's what I can tell you:

PCR is successful... lots of amplification, even after gel purification.

I ran a gel of my DIGESTED samples to make sure I didn't lose them in some ethanol precipitation step or something... I ran the gel to ensure that, post digest, I still had pHTT7K vector and each of my M2, M3, M4 inserts... the gel showed I had lots of each.

After ligation, and after electroporating and plating, I get a decent amount of colonies growing on the plates... about the same as M1... but when I grow up broth cultures of colonies NONE of them have my insert in them! Since the colonies grew, obviously it's not a problem in electroporating or bad competent cells. So, something must be going wrong with my ligation (I assume).

Now, the only thing I can think of is maybe I'm not using enough insert. I know how to calculate the ratios, but the thing is is that since the inserts are so small, and the samples I have run at about 100ng/uL, I'm only adding a tiny volume of my insert samples to the ligation mix (on the order of 1uL of M2 to 0.5uL of M4).

If anyone can help me with this and give some suggestions, it would be much appreciated... it's really strange to me how I have lots of digested insert and vector, and yet no positive clones at all after successful transformation and growth.

Hopefully, with help, I'll get this figured out!

Lanyon

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It appears to me that something went wrong with your ligation.

I would prefer to do it again. I would gel extract my PCR products as well as vector rather than PCR clean up or ethanol precipitation.

You can calculate the ratio although I am not doing that now (simply look at the band density on gel and roughly add "appropriate" amount of PCR products), and always get my ligation worked.

You got lots of colonies that may be due to vector self-ligation or some very small DNA fragments from your PCR ligated to vector (that's why I would suggest you to gel extract the target PCR). So you can either try the above suggestions or check your vector though vector should be ok since you have just done successfully with M1.


Try again and goodluck

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Thanks for the reply, but I just forgot to mention one thing...

Although I did not do it for M3 and M4, I DID try gel-purifying BOTH the vector and insert for M2 for my second attempt, and it still didn't work. Sorry i didn't mention it before. Still funny though, seeing as how I did not have to do this for M1.

Lanyon

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Just a simple question what's the length between the end of your insert and the restriction site ? Remember that for SacI you need only 1 base extra to cut
[b]but for NdeI you need at least 7 base pair on the outside of your primer and it's listed to cut only 75% in two hours (Biolabs catalog Restriction enzyme properties table)

So if the length is not the same between M1, M2, M3 and M4 that might be an explanation !

What you can do is cloning your fragment in a pGEM-T or Topo vector and the cut out inserts with your enzymes it's another step but sometimes the quickest way to sucess is not the straight one

Pesji

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I agree -- whenever we need Nde I ends on a PCR product (for example), we'll include the restriction site as a 5' addition to the primer(s), TA clone the product, and liberate it with Nde I.

This comes up surprisingly often in our lab when we're making N-terminal his-tag fusions; the intermediate TA cloning step has become routine in our lab when dealing with this enzyme...

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