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Restriction analysis of a vector - (Oct/03/2005 )

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I've been trying to learn basic molecular cloning techniques. I have prepared competent cells of E. coli JM101 and HB101 and then transformed them with pUC18 vector. The transformants were screened for ampicillin resistance. After that I did both mini and maxprep alkaline lysis method to isolate plasmid. Using 1% agarose gel, I could detect two bands of the plasmid as expected but through the marker used which was Lambda DNA/HindIII I could see the 2 plasmid bands were below that of 2322 and 2027 bps of the marker. The size of pUC18 is 2686 bps. So is this expected?

Next I've been doing restriction analysis of standard pUC18. My setup is as follows:

HindIII (5U/uL) >>>>>>>>>>>>2 uL
pUC18 (10ng/uL)>>>>>>>>>>>6 uL
Restriction buffer (10X) >>>>>>2 uL
Sterile distilled water >>>>>>>>10 uL

Then I incubated this restriction mixture at 37 degree celsius for 4 hours. Then the restriction is quenched by heating at 65 degree celsius for 20 minutes. After this I stored it at -4 celsius. Next day I took 8 uL from it, mixed 1uL of EcoRI and 1 uL of its buffer. This restriction mixture was incubated at 37 for 4 hrs and then quenched at 65 for 20 minutes.

I did agarose gel electrophoresis using 1% gel of both taking loading dye and the sample in 5:5 ratio but could see no bands.

I have my doubts over the setup. I am using 10 U of enzyme for 60 ng of plasmid. Is that where the problem lies? Am I using too much of the enzyme? I guess too much of enzyme is suicidal as far as success is concerned. It would be great for me a novice in this field if you people could throw some light upon it.

Best Regards,
Rigien

-rigien-

I know that it use 1-10 U of enzyme for 1 ug di DNA, I think you use too much enzyme.

Best regards

-ryu00-

you used too little DNA (only 60 ng in total). you can use 10 times more next time.

-bullfrog-

Actually, there was only 24 ng total in the final sample (8 ul of a 3 ng/ul solution). I suspect that's why you didn't see it.

By altering your digestion procedure a bit, you can save a lot of time and money:

  • 10 units of enzyme is way too much -- 1 to 4 units is plenty.
  • You can likely cut with EcoRI and HindIII at the same time in a double digest -- check your enzyme supplier's buffer chart for a buffer in which they're both compatible.
  • There's no need to digest for four hours -- two is usually plenty, most times I digest for just one hour.
  • I do not find it necessary to heat-inactivate a restriction digest at all, especially if I'm going to run the sample out on a gel as the next step.
As to your other question, running a sample of undigested plasmid DNA is expected to produce three bands corresponding to the three forms of the plasmid -- supercoiled circular (smallest band), nicked circular (middle band), and linear (largest band).

Good luck!

-HomeBrew-

just to add a precision to the very good post of Homebrew the supercoiled DNA migrate faster than the linear so it always appear smaller than his normal MW size cool.gif

pesji rolleyes.gif

-pesji-

don't forget to stain with ethidium bromide also.

-tap14-

I just happened to be in this situation, too. EcoR I and Xba I (or Pst I) were used to double digest
the vector of pET32a at 37 degree celsius for 2 hours and then I ran the sample out on 1% gel, only the plasmids but no target bands were seen. I have repeated four times but got the same results.

-geness-

geness -- do you mean you saw linearized plasmid, or did you see three bands as described above? One (a single band) indicates a failed cloning reaction, the other (three bands) indicates a failed digest. Which pattern did you see?

-HomeBrew-

Thank you all for the responses.

But I still can't figure out why the pUC18 vector didn't undergo restriction. I had 60 ng of the vector cut with 10 units of Hind III. So will the use of less unit of the enzume result in a successful restriction? Where I am studying as an udergrad which is in Nepal we can't afford to buy much of these expensive chemicals. So is there any way I can have a sucessful restriction using just around 60 ng and not as bullfrog suggested the use of 10 times more. One more thing I heard somewhere that restriction enzymes are not to be thawed. Is that true? If yes then how am I suppose to work with them?

Best Regards,
Rigien

-rigien-

QUOTE (rigien @ Oct 5 2005, 05:27 PM)
Thank you all for the responses.

But I still can't figure out why the pUC18 vector didn't undergo restriction. I had 60 ng of the vector cut with 10 units of Hind III. So will the use of less unit of the enzume result in a successful restriction? Where I am studying as an udergrad which is in Nepal we can't afford to buy much of these expensive chemicals. So is there any way I can have a sucessful restriction using just around 60 ng and not as bullfrog suggested the use of 10 times more. One more thing I heard somewhere that restriction enzymes are not to be thawed. Is that true? If yes then how am I suppose to work with them?

Best Regards,
Rigien


Well the problem is clearly that you don't have enough DNA so you can't visualize 60ng at the beginning but after your digest much less cause you didn't use all of the first digest mix !

The limit is around 20ng for one single band and in a very well prepared gel with TBE !

If you start with 10 times more DNA as suggested you will surely see your product, they might be a possibility of incubating directly with both enzymes check the NEB catalog you will find in the appendix a chart named "Double digests" check in which buffer you can combine your enzyme.

Enzyme definition is
1 unit cuts 1 microgram of DNA in 1 hour

So for 600 ng you need theoretically only 0,6units since most enzyme are delivered as 10units / microliter I'm sure that using 0.5 microliter of each enzyme is more than enough.

Yes the enzymes are heat labile that means you should keep them on ice when you use them.

So to summarize
Take approximately 600ng of DNA add 2microliters of 10X concentrated restriction buffer, 0.5 microliters of each enzyme (HindIII and EcoRI) and adjust the total volume to 20 microliters with DD H2O. Incubate for 2 hours at 37°C and run the entire digestion on a 1.5% Agarose gel made in 0.5xTBE

This should work fine !

pesji cool.gif

-pesji-

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