A cloning nightmare - (Sep/02/2010 )

Hi all

This is my first post so please excuse (1) the lengthiness and (2) possible inaccuracies.

I have been trying to clone the FLI1 gene for months using the pCAG vector system. I have successfully managed to clone two other genes into this vector ~March time, however when starting with FLI1 I have had incredible trouble. I'll list my main problems below.

Achieved so far:

1. I have isolated the FLI1 gene from a cell line through RT-PCR and performed a restriction enzyme digest to confirm it is in fact the correct thing.

2. Cut open the pCAG vector at appropriate restriction sites matching the sites I designed my primers with for PCR of FLI1 (XhoI and NotI)

3. Attempted to ligate FLI1 (extracted from a gel using Sigma's gel extraction kit) to pCAG-empty vector (also extracted using the same kit).

4. Transformed XL1 blue competent cells with the ligation product and left to grow on Ampicillin+ agar plates.

5. Pick colonies and perform a miniprep.

6. Restriction enzyme digest all minipreps with XhoI and NotI to ensure FLI1 is indeed in pCAG

...... It isn't!

I have tried the above through twice and got the same results both times. FLI1 just is not in the vector after the miniprep. The vector is certainly there as the XL1 cells are growing on ampicillin plates (pCAG harbours the AMP resistance gene).

Advice I have been given: "Try adding CIP to the cut vector to stop it from self-ligating"

And so I tried this. However, here comes my next problem:

After extracting cut pCAG from a gel, obtaining a good DNA yield, adding CIP for 1 hour at 37oC, I then go to load it onto a gel once more. As SOON as the DNA touches the TBE the gel is held in it floats out of the well like a ghost. I have been told this may be due to the presence of ethanol in the DNA. However, I follow the Sigma kit's protocol exactly. This consists of a wash step with ethanol spun through a column for 1 minute at 13,000 g. Ethanol is emptied and the column is spun again with no ethanol added for another 1 minute at 13,000 g. Surely this is enough to remove the ethanol?

These are pretty much all my problems summed up so far. Funnily enough when cloning my first two genes I encountered no problems at all, but with FLI1 I seem to have hit every barrier possible. That's science I guess. If anybody has any suggestions or help they can give me it would be greatly appreciated. I'm open to pretty much anything right now.

Cheers!

Neither XhoI nor NotI are very good cutters near the end of PCR products, particularly NotI (see here). When you designed your primers, how many irrelevant 5' bases did you add upstream of your restriction site?

I don't think it's going to matter, however, because I don't think you can add enough to satisfy NotI in particular. So, in a case like this, I would TA clone my PCR product first, then release it from the TA vector using NotI and XhoI, recover *that* fragment, and clone it into your ultimate vector.

Thanks for your reply.

Interestingly I haven't really had any problems with XhoI and NotI cutting. How do you think this could be affecting the problems I am experiencing? As for redundant bases, I barely put any... maybe three in the primer design.

I'm not sure what you mean by TA clone. I do, however, have a vector called pGEM-T Easy (not used it yet). The product description reads as so:

"T-Overhangs for Easy PCR Cloning: The pGEM®-T and pGEM®-T Easy Vectors are linearized vectors with a single 3 ́-terminal thymidine at both ends. The T-overhangs at the insertion site greatly improve the efficiency of ligation of PCR products by preventing recircularization of the vector and providing a compatible overhang for PCR products generated by certain thermostable polymerases".

I believe it also works in a 'rapid ligation' way, in that you leave it with your insert for an hour at RT and it combines. Also, the insert is flanked by EcoRI sites for easy removal.

Is this the kind of thing that you mean?

Pardon my ignorance, but I do not understand why I would want to put my insert into this vector, only to cut it out again and once again try to ligate it into the pCAG vector?

Thanks again!

Ok I will attempt to answer my own question. It's 1am and I don't think I have much chance of sleeping until I have this straight in my head!

The FLI1 gene which I have RT-PCR'd is flanked with XhoI and NotI with very few redundant bases on the ends. According to the link you gave me, hardly any XhoI and NotI will be cleaved due to the low numbers of flanking bases. Also, I only cut for 2 hours at a time. When cutting out the trimmed FLI1 after the RE digest, most of the band will be full of product that has not been cleaved, and as such, uncleaved product will not ligate into the vector.

Am I right?

I will carry on...

If I clone into a TA vector, XhoI and NotI will now be flanked with many bases meaning they can be cleaved successfully leaving FLI1 ready to ligate into cut pCAG.

I hope to god I'm right because I could do with some sleep now.

Cheers!

Yep -- you got it. The enzymes you're using have a quite difficult time cutting when the sites are close to the ends of a linear fragment, such as the PCR product you're trying to clone. This is likely the cause of your difficulties -- your vector is probably fine, but your insert is undigested, thus it doesn't have the the ends necessary for successful ligation. Taq polymerase adds adenosine residues to the 3' ends of your PCR product, thus your PCR product is double stranded but with a single A overhang on each 3' end. A TA cloning vector has 3' thymine overhangs, so your PCR product can be cloned into such a vector without digestion.

Once your product is cloned into the TA vector, the XhoI and NotI sites you engineered into your primers are flanked on either side by the whole expanse of the vector and insert DNA, thus their reluctance to cut near the ends of fragments is nullified, and you can recover your fragment from this clone without difficulty, and be assured that your insert now has the correct overhangs for cloning into your ultimate vector.

Get some sleep -- and good luck!

Hi
I don't have too much to add to what Hemobrew have already told you - just a small advise.
You may add CIP directly into restriction reaction after heat inactivation and then purify your digested vector on gel.
Extra purification step is not necessary.
Good luck
Michael

andyg2886 on Fri Sep 3 00:08:08 2010 said:

Homebrew - thanks for your advice and explanation, it's help like this that gets missed in my lab as I'm the only person ever to have tried cloning. I'm very greatful for your help, and will keep you updated with my cloning work.

Michaelro - I had never thought of that before, but your advice certainly makes sense. If I'm right in thinking, I can heat-inactivate my enzymes at 65oC for 20 minutes. Add CIP directly to the RE mix?

Another question, however, it still remains that I need to gel extract after treating with CIP. Performing this step does not negate the possible carry-over of ethanol in the gel extraction step. Once this step is done I need to run the digested and CIP'd product on a gel and after extraction, any ethanol present in the mix would be detrimental to further work, surely?

I am going to give both ways a try, firstly because Homebrew - the evidence about XhoI and NotI looks convincing. However, I will continue with the other way too as I have already successfully cloned two other genes with XhoI and NotI before with no problem.

If you have any advice on the ethanol carry-over, how to stop it, or how to remove it from the final sample, that would be much appreciated.

Cheers and thanks again

andyg2886 on Fri Sep 3 09:54:34 2010 said:

A two step elution guarantees that the column sees low ethanol concentration on the second spin, releasing DNA even if the first elution has excess alcohol. Using the DNA in low volume compared to the reaction volume in a subsequent reaction dilutes the ethanol to low levels.