Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Restriction enzyme digest


  • Please log in to reply
19 replies to this topic

#1 xyz74

xyz74

    member

  • Active Members
  • Pip
  • 8 posts
0
Neutral

Posted 27 January 2009 - 12:13 PM

I am doing a subcloning experiment and have some questions about the digest.

My insert is in EcoRI and XhoI. My vector has EcoRI and XhoI sites but they are only 5 base pair apart. The vector also has SalI sites. The NEB catalog shows that XhoI and SalI have compatible ends.

So my question is can I digest the insert with EcoRI and XhoI and the vector with EcoRI and SalI and then ligate.

Thanks.

#2 swanny

swanny

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 367 posts
8
Neutral

Posted 27 January 2009 - 06:34 PM

I am doing a subcloning experiment and have some questions about the digest.

My insert is in EcoRI and XhoI. My vector has EcoRI and XhoI sites but they are only 5 base pair apart. The vector also has SalI sites. The NEB catalog shows that XhoI and SalI have compatible ends.

So my question is can I digest the insert with EcoRI and XhoI and the vector with EcoRI and SalI and then ligate.

Thanks.

The idea sounds OK.
Keep looking through the NEB website. There will be pages that tell you about how efficient different enzymes are close to the ends. You might need to one, clean up, then do the other.
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.

#3 perneseblue

perneseblue

    Unlimited ligation works!

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 578 posts
17
Good

Posted 27 January 2009 - 06:43 PM

yes, you certain can. :angry:

However please note there have been problem with SalI ligations. More frequently than expected and for reasons unknown, people encounter problems when using SalI in their ligation strategy. So be advised of potential ligation difficulties.
May your PCR products be long, your protocols short and your boss on holiday

#4 swanny

swanny

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 367 posts
8
Neutral

Posted 27 January 2009 - 07:47 PM

yes, you certain can. :angry:

However please note there have been problem with SalI ligations. More frequently than expected and for reasons unknown, people encounter problems when using SalI in their ligation strategy. So be advised of potential ligation difficulties.

Yes, definitely go to the SalI page in NEB.com for their FAQs.
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.

#5 scolix

scolix

    a student

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 314 posts
5
Neutral

Posted 01 February 2009 - 07:23 AM

As already pointed out, you may have problems with salI with some plasmids and some it works like a charm.

#6 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 03 February 2009 - 10:28 PM

Hi everyone, I just wanna ask how long do you guys usually leave your enzyme digestion? Is 3 hours enough?
Has anyone tried making mutated libraries using error-prone PCR and doing an enzyme digestion on the PCR products so they can be cloned into vectors?

Thanks in advance.

#7 hanming86

hanming86

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 156 posts
2
Neutral

Posted 04 February 2009 - 03:28 AM

Hi everyone, I just wanna ask how long do you guys usually leave your enzyme digestion? Is 3 hours enough?
Has anyone tried making mutated libraries using error-prone PCR and doing an enzyme digestion on the PCR products so they can be cloned into vectors?

Thanks in advance.


3 is good enough .

regarding the mutated library ... i think it's quite a nasty business. as you do not know where the error will occur and it might occur more than once albeit rarer.

However, it definitely depends on some factors such as how long the PCR product is , how error-prone is the polymerase so on so forth.
Lab + Coffee + Music = Bliss

#8 scolix

scolix

    a student

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 314 posts
5
Neutral

Posted 04 February 2009 - 03:32 AM

I would leave it for an hour. It should be plenty of time.

You could try site directed mutagenesis instead of error prone PCR.

#9 rkay447

rkay447

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 180 posts
20
Excellent

Posted 04 February 2009 - 05:19 AM

I did something similar in that I random mutagenized the gene by doing a gene specific pcr and then ligating into the vector. You would need to use primers that are specific to the vector your library is currently in since there are thousands of different open reading frames. Not sure why you would do this since libraries are used to isolate single genes or screen for specific genes. You then mutate the single cDNA you isolated in whatever experiment or screen you preformed (what I did). But, it can be done. Do the pcr, dpn1 digest the original template library, digest with your restriction enzymes, pcr purify or precipitate (note that you will products of many different sizes so gel purification won't work), ligate. Problem is you will most likely loose a number of cDNAs. Why not attempt "round the world" pcr where you amplify the entire vector using taq? This way each cDNA gets amplified and hopefully mutated. You just treat the pcr with dpn1, transform and amplify the mutated library.

We did this to try to identify binding mutants. We had no clue where to place a site-directed mutagenesis so we went on a fishing-expedition and just randomly mutated the gene. Did two hybrid screens and identifed clones that interacted the way we wanted. Isolated the cDNA and sent for sequencing to identify where the mutation occured. Mine had two mutations which we then tested individually by doing site-directed mutations. Managed to identify a single residue critical for interaction and function in the protein of interest.

Edited by rkay447, 04 February 2009 - 05:24 AM.


#10 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 04 February 2009 - 06:28 AM

Hi everyone, I just wanna ask how long do you guys usually leave your enzyme digestion? Is 3 hours enough?
Has anyone tried making mutated libraries using error-prone PCR and doing an enzyme digestion on the PCR products so they can be cloned into vectors?

Thanks in advance.


3 is good enough .

regarding the mutated library ... i think it's quite a nasty business. as you do not know where the error will occur and it might occur more than once albeit rarer.

However, it definitely depends on some factors such as how long the PCR product is , how error-prone is the polymerase so on so forth.


Yes, i think it is a nasty business trying to produce a mutated library.I have been having lots of problems getting a mutated library and I am supposed to use error-prone PCR. Just a few days ago, I finally optimized my PCR in such a way that I got the PCR products I want. FYI, I have not been getting the PCR products that I want so it was a pleasant surprise.

Treated it with Dpn1 and did a reamplification after that. I did a double digestion on my PCR products. When I ran it on a gel, the band was quite faint. This is seriously annoying because I need to use the digested PCR products for my ligation. Funny thing is, though the band for my digested PCR products are very faint, I can get high concentrations of the digested PCR products. The highest I got was over 200ng/ul, which was a surprise. What usually happens when I run a gel after enzyme digestion is that the band can become so faint that it can barely be seen unless I overexpose the gel. I am not too sure why this happens. :ph34r:

#11 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 04 February 2009 - 06:29 AM

I would leave it for an hour. It should be plenty of time.

You could try site directed mutagenesis instead of error prone PCR.


Hello,

Thanks for your reply. Would it be better to do a double digestion at the same time, or is it usually better to do a sequential digestion with PCR cleanup in between?

#12 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 04 February 2009 - 06:35 AM

I did something similar in that I random mutagenized the gene by doing a gene specific pcr and then ligating into the vector. You would need to use primers that are specific to the vector your library is currently in since there are thousands of different open reading frames. Not sure why you would do this since libraries are used to isolate single genes or screen for specific genes. You then mutate the single cDNA you isolated in whatever experiment or screen you preformed (what I did). But, it can be done. Do the pcr, dpn1 digest the original template library, digest with your restriction enzymes, pcr purify or precipitate (note that you will products of many different sizes so gel purification won't work), ligate. Problem is you will most likely loose a number of cDNAs. Why not attempt "round the world" pcr where you amplify the entire vector using taq? This way each cDNA gets amplified and hopefully mutated. You just treat the pcr with dpn1, transform and amplify the mutated library.

We did this to try to identify binding mutants. We had no clue where to place a site-directed mutagenesis so we went on a fishing-expedition and just randomly mutated the gene. Did two hybrid screens and identifed clones that interacted the way we wanted. Isolated the cDNA and sent for sequencing to identify where the mutation occured. Mine had two mutations which we then tested individually by doing site-directed mutations. Managed to identify a single residue critical for interaction and function in the protein of interest.


Hello rkay447,

Thank you for the reply.

The main reason why I am doing an error-prone PCR, Dpn1 digestion, followed by a reamplification is because I am producing a mutated library for selection purposes.

I am provided with a fusion gene, which is linked to pET22b and I am doing an error-prone PCR to get mutated versions of that fusion gene. Those are the ones which I will be using for a selection, which is why I am not doing a site-directed mutagenesis.

And also, you mentioned "note that you will products of many different sizes so gel purification won't work". What does this mean? Plus, is PCR cleanup or gel purification better? From what I know, PCR cleanup cleans the small pieces of wanted DNA, but it does not necessarily give you the digested PCR products that you want to ligate to your digested vector (which can be achieved by gel purification). Plus, if you get other bands in addition to the desired band that you want, won't PCR cleanup still give you your digested PCR products together with products present in other bands? :ph34r: I hope I am not confusing you here.

Do advise me. Thanks in advance!! ;)

#13 rkay447

rkay447

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 180 posts
20
Excellent

Posted 04 February 2009 - 09:08 AM

Ok, I think there is a little confusion here because of the term library being used incorrectly. A library refers to a mixture of cDNAs representing every (or most) of the genes being expressed at a single moment in time in an organism, tissue, cell line, ect. You take a sample of whatever you want to make the library from (cell lines, xenopus embryos, tissue sample, ect.) and run a reverse transcriptase reaction where every single mRNA present in the sample is reverse transcribed to DNA. This is then ligated into a vector to give the mixture of cDNAs. There are thousands of genes in the one sample which are all different sizes. This is why I was thinking you would get products of all different sizes. If you do a vector specific primer pcr of a library, you are going to amplify all of the thousands of genes which all have different sizes and therefore can not be purified by a gel.

However, it sounds like you are working with one gene and are trying to mutate the one isolated cDNA. Yes? Then I highly recommend you gel purify the pcr to eliminate the template plasmid, digest and pcr purify to remove enzymes and buffer. Quantify and ligate.

Edited by rkay447, 04 February 2009 - 09:10 AM.


#14 scolix

scolix

    a student

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 314 posts
5
Neutral

Posted 05 February 2009 - 07:50 AM

I would leave it for an hour. It should be plenty of time.

You could try site directed mutagenesis instead of error prone PCR.


Hello,

Thanks for your reply. Would it be better to do a double digestion at the same time, or is it usually better to do a sequential digestion with PCR cleanup in between?


If you are using compatible buffers, then I would do a double digest. Saves time.

#15 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 06 February 2009 - 05:01 AM

Ok, I think there is a little confusion here because of the term library being used incorrectly. A library refers to a mixture of cDNAs representing every (or most) of the genes being expressed at a single moment in time in an organism, tissue, cell line, ect. You take a sample of whatever you want to make the library from (cell lines, xenopus embryos, tissue sample, ect.) and run a reverse transcriptase reaction where every single mRNA present in the sample is reverse transcribed to DNA. This is then ligated into a vector to give the mixture of cDNAs. There are thousands of genes in the one sample which are all different sizes. This is why I was thinking you would get products of all different sizes. If you do a vector specific primer pcr of a library, you are going to amplify all of the thousands of genes which all have different sizes and therefore can not be purified by a gel.

However, it sounds like you are working with one gene and are trying to mutate the one isolated cDNA. Yes? Then I highly recommend you gel purify the pcr to eliminate the template plasmid, digest and pcr purify to remove enzymes and buffer. Quantify and ligate.



Hi there,

I am sorry about the confusion. Yes, I am working with one gene, which is a fusion gene, and I am trying to mutate it so I can get a lot of copies of variants.

What I did was to digest my PCR products and gel purify the band which I wanted. Would it have any difference from gel purifying the PCR products first before digestion and doing a PCR cleanup? My supervisor told me that the advantage of a gel purification after digestion is that you definitely get the product that you wanted.

I did my ligation and did a PCR on the ligated product to see if ligation was successful. Ran a gel, but there were smears and a lot of unspecific bands, included the band for self-ligated vector. My gene library band is there, but I have no idea why there are so many other bands along with it. :lol: Now, what I am trying to do is to rescue the library. I have done a transformation with the ligated products and will be doing a miniprep to prepare it for sequencing to see if I get the inserts in my vector and whether the vectors are mutated.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.