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how do you check your restriction enzyme is working? - (Oct/12/2005 )

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i will digest my vector with NotI and XbaI. I did single digestion for my vector, and both showed single band. then I think the enzyme is ok, then I did double digestion with NEB 3 and BSA overnight and also my PCR clean up product also digest overnight. next day i run then digested product on gel, they are all single band and I cut off bands, do gel extraction, ligation, transformation. finally I found my negative control which is only vector got colonies blink.gif . I even did dephosphorylation after i digest. I am totally lost.
I am thinking it is possible one of the enzyme no working at all. how could you tell whether your enzyme is working then

Thanks a lot
I did this for two weeks now. drive me mad

Thanks millian
cathy

-cathy-

I am confused. should I see three bands after single digestion?
if so what is these three bands are?

-cathy-

Hi,
I agree with what you suspect. The problem may on the vector, or PCR product (insert), or even both.
Could you first insert your PCR product (withour RE cutting) into some T/A cloning vector such as pGEM-T Easy? Then you cut off your insert to make sure that your insert got no problem on both sides.
If you still don't get any correct colonies, you need to further check your vector:
1. Are the two RE sites that you are going to use too close to each other? If yes, try sequential digestion, i.e. heat inactivate the first Re before adding the next
2. Is that possible for you to change an alternative, but comparable cutting site? e.g. XbaI vs. SpeI; BamHI vs. BglII

good luck smile.gif

-bullfrog-

Hang in there dude, I have been there before with things like this and I know it is frustrating. The first thing to do (and it sounds like you are doing it) is to go back to the basics. Start with the plasmid maps and the sequence of your pcr product and make sure the restriction sites are there. Secondly, I recommend at this point that you first clone your pcr product into a topoTA cloning vector (invitrogen) so that you can verify that you are getting good cutting of the ends of your insert. Third, drop the dephosphorylation step, it makes no sense here and may actually hide what is really going on).Fourth, bump-up the ratio of insert to vector that you include in your ligation and buy a new T4 ligase (The ATP can go bad sometimes after repeated freeze-thaw cycles). If all this does not work, come back and we can try to figure out what else you can do. Good luck and fight through this, IT SHOULD WORK!

ps XbaI is only 75% efficient in buffer 3, maybe try a sequential digest, it may remove some of your background!

-tap14-

Are your restriciton sites in your primers, and if so: how many extra bp have you added? Some sites advise 6 bp, but you better have at least 10 or so. Might be safer indeed to clone into a TA cloning vector, and then cut that one (as tap14 states, sequential) and gel-purify your needed product.

-vairus-

Even with an overnight double digestion you'll probably still have some uncut plasmid.

Don't worry about it. That's why we screen recombinants.

-Matt

-MisticMatt-

QUOTE (cathy @ Oct 12 2005, 09:09 AM)
i will digest my vector with NotI and XbaI. I did single digestion for my vector, and both showed single band. then I think the enzyme is ok, then I did double digestion with NEB 3 and BSA overnight and also my PCR clean up product also digest overnight. next day i run then digested product on gel, they are all single band and I cut off bands, do gel extraction, ligation, transformation. finally I found my negative control which is only vector got colonies :blink: . I even did dephosphorylation after i digest. I am totally lost.
I am thinking it is possible one of the enzyme no working at all. how could you tell whether your enzyme is working then

Thanks a lot
I did this for two weeks now. drive me mad

Thanks millian
cathy


Dear Cathy,

There are some tips that may be helpful in your cloning. When you do single digestion, always run the digested vector DNA along with the undigested one under the control of DNA ladder - sometimes undigest vector may look like a linearized DNA, but size will always be different from the one indicated on the map. After making sure that the enzymes do cut, run low scale double digest (up to 30 uL) to check whether the NEB buffer you picked satisfies both REs.

Double restriction digest (RD) should give you two bands (if the enzymes are single cutters). Check the size of your band and cut it out, but remember, to clean up your vector completely out of undigest DNA, you may have to run it for more than 2 hours at 80-100W (depending on the size of your vector). Failure to do that will result in quite a few clones on your empty vector plate.

It is always a good idea to use a TOPO vector as a vehicle for your PCR products you want to clone.

Good luck

-cska_fan-

Thanks folks
that is really nice to have you guys here. I did add extra bp at the both end of my primers for PCR. I did concern about the distance between Xbal and noti in my vector, It is only about 10bp. Because of this, I am not able to the cutted band from my vector, that is the problem. So I am thking to do a sequetial digest rather than double digest, maybe help.
By the way, I did not use the T/A cloning vector, any more info about it?

-cathy-

QUOTE (cathy @ Oct 13 2005, 11:03 AM)
Thanks folks
that is really nice to have you guys here. I did add extra bp at the both end of my primers for PCR. I did concern about the distance between Xbal and noti in my vector, It is only about 10bp. Because of this, I am not able to the cutted band from my vector, that is the problem. So I am thking to do a sequetial digest rather than double digest, maybe help.
By the way, I did not use the T/A cloning vector, any more info about it?



hi Cathy,

here is the link
https://catalog.invitrogen.com/index.cfm?fu...Description=758

good luck

-cska_fan-

In a sequential digest, you digest with one enzyme in a lower salt concentration and then bump up the salt concentration to comply with the requirements for the second enzyme. If you look at the buffer components you will see the differences in the composition of the different enzyme buffers. In your case I belive you should start with the XbaI buffer and then increase the salt for the second enzyme. In most cases it is not necessary to inactivate the enzyme of repurify the DNA. After the second digestion you should be good to go.

-tap14-

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