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problem with cloning/restriction digest

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problem with cloning/restriction digest

Postby biosci02 » Fri Jan 25, 2008 12:49 am

I am trying to clone an insert into a Pet-30 vector. I generated the insert by PCR adding on NdeI/XhoI ends. I cleaned up my pcr using the Qiaquick PCR purification. I digested my product (using Nde/XhoI) for about 3 hrs @37 deg. When I run the digest on a gel, I see my insert (which appears to be the right size), but not the vector. Help?
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Postby canalon » Fri Jan 25, 2008 12:53 am

Wait if I understand you successfully ligated your insert in pET-30 and transformed it in bacteria, but when you are running minipreps to check if you have your construction, you see only the insert? That is really strange.
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Re: problem with cloning/restriction digest

Postby biosci02 » Fri Jan 25, 2008 1:22 am

Thanks for responding. I didn't explain very well. I'm trying to do restriction stick end cloning. I've added the restriction enzyme target sites (NdeI/XhoI) on my insert and afterwards I purified the PCR product with the QiAquick kit. Then I did the digestion with the enzymes. I was hoping to get an insert I could cut out, gel purify and then ligate into a Pet-30 vector cut with Nde/XhoI. I hope I've explained better.
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Re: problem with cloning/restriction digest

Postby blcr11 » Fri Jan 25, 2008 9:19 am

I don’t understand either. There shouldn’t be any vector to see, if I understand you correctly. You amplified a fragment by pcr, adding the required ends in the process. You then purified the product and digested with restriction enzymes. There is nothing to “cut out” that I see. It’s already out. You just need to ligate the digested fragment into NdeI/XhoI digested pET30, right? Or am I missing something? Unless you're saying that your vector digestion (which should have been a separate digest at this point) failed for some reason.
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Re: problem with cloning/restriction digest

Postby blcr11 » Fri Jan 25, 2008 7:41 pm

Just so I'm being clear: after you digest your pcr fragment you probably want to purify the now-digested fragment on an agarose gel, but you shouldn't see any vector DNA on the gel--not in the same lane, anyway. This step is to remove any possible interference from the RE digest biproducts (especially the free nucleotides) before ligation. You can sometimes get away with a simple phenol extraction/ethanol precipitation step instead since the smaller nucleiotides don't precipitate as efficiently as the larger oligonucleotide, but usually you do a gel purification to be safe. You also digest a few micrograms of pET30 with NdeI/XhoI and purify the digested vector on an agarose gel. The fragment and the vector will be run in separate tracks at this stage of the process.
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Postby canalon » Sat Jan 26, 2008 12:08 am

Yep you are doing fine, just missing the final steps: purify your fragment as blcr11 said (I think you can use some kits like QiAgen PCR purification kits if you are rich and do not want to run the gel) Digest your vector without insert and purify. Mix both purified vector and insert in your favorite ligation mix and transform in E.coli by heat shock or electroporation. Give the cells 1h in SOC to recover and then plate on selective medium . If everything went well you should now see plenty of colonies on your plate and you can miniprep some of them to see if they all inserted the right construct.
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Re: problem with cloning/restriction digest

Postby ashlie23 » Thu Feb 07, 2008 11:10 pm

Guys, I am new to this forum so I am probably not supposed to ask question under this thread but since this is regarding the similar question I have, perhaps some of you can help me with this!

I am using pTYB2 expression vector (7.4 kb) and doubly digesting with Nde and Xma to produce sticky ends. The insert DNA has also been digested with same enzymes. I have been having some major problems with my cloning:

(1) I am unable to GEL PURIFY the digested vector. Meaning that I am only getting <10% of the DNA I start out with when I set up the digest. Digested insert DNA is not a problem. I get pretty good concentration after gel purifying.
I have tried this so many times with different vector conc. to begin with - followed each step of the SV Wizard Promega Gel cleanup protocol very carefully. Others also recommended phenol chloroform extraction w/o running on a gel but that would not get rid of circular/supercoiled vector DNA. And in return, either I am not getting ANY colonies or a lot on control as well as vector + insert which doesnt carry the insert but only an uncut vector.

Does any one have any idea on what else I could do? I have used this vector before and even gel purified it, but this time it seems like causing such a problem. Please let me know if I can give any more info in order to be better assisted.

Thanks a lot in advance.
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Re: problem with cloning/restriction digest

Postby blcr11 » Fri Feb 08, 2008 3:38 pm

If you are seeing significant amounts of supercoiled and/or relaxed vector in your digests, then your enzyme(s) isn’t cutting very efficiently—or are you just worried about any residual uncut vector DNA? Is your vector DNA prep really old? Maybe you just need to make a fresh prep of vector DNA—or buy some more, though NEB seems to have discontinued the old IMPACT kit. I don’t know if the old vectors are still available. Or maybe you need to do the phenol extraction before you do the digestion on the assumption that an old prep has accumulated some kind of inhibitor. Another—remote—possibility is methylation. NdeI doesn’t care about methylation, but XmaI is inhibited by CpG methylation. But the methyl transferases responsible for CpG methylation are eukaryotic enzymes, not bacterial so far as I know. I guess this isn’t a very satisfying solution and it may not work (or you’ve tried it already), but if it were me, I would make or buy fresh pYTB2 vector, get fresh NdeI and XmaI, and do it again.
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Postby canalon » Fri Feb 08, 2008 7:22 pm

What I am not sure to understand:
1- You start with some purified circular plasmid,
2- you (double) digest it
3- you load it on gel, and cut the band corresponding to the digested portion
4a- you load a small sample of the purified vector on a gel to check the quantity/quality before ligation
OR
4b-you measure OD to estimate DNA concentration after gel extraction

When you say that your gel purification does not work, you have compared DNA concentration at step 4(a or b) with step 1 or with the band of cut vector on step3 ?
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Re:

Postby ashlie23 » Fri Feb 08, 2008 8:15 pm

CANALON, thanks so much for your reply. Here's my info:

Yes! (1) I start with - let's say - 2 to 4 ug DNA to be digested.
(2) Dbl digest the vector with Ndel and Xma I (at the same time I also set up single digest side by side as a control)
(3) All samples including dbl digested vector and singly digested are loaded on 1% gel. Then the bands corresponding to the linear vector is excised and subsequently purified by promega/qiagen/eppendorf Gel purification kit
(4) The eluted DNA measured on Nanodrop and I get less than 5% back compared to what I start out with when setting up a digest.( I understand that only linear band is excised and thus genomic/chromosomal DNA will not be present when u excise the band and the concentration of DNA to begin with represents both genomic and plasmid DNA). But here I am not getting anything back.
- I've also run the digested and purified vector DNA on a gel to see whether it shows up on the gel and I see a faint band where it should be (~7.4kb) but subsequent ligation with this vector does not give any colonies.

I have tried gel purifying DNA with different kits. Like I said before, PCR and digested insert DNA purifies just fine (200-900 bp) but its the digested vector DNA that's so hard to gel purify. I hope this will help answer my question. If more info is needed, please let me know.


canalon wrote:What I am not sure to understand:
1- You start with some purified circular plasmid,
2- you (double) digest it
3- you load it on gel, and cut the band corresponding to the digested portion
4a- you load a small sample of the purified vector on a gel to check the quantity/quality before ligation
OR
4b-you measure OD to estimate DNA concentration after gel extraction

When you say that your gel purification does not work, you have compared DNA concentration at step 4(a or b) with step 1 or with the band of cut vector on step3 ?
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Re: problem with cloning/restriction digest

Postby blcr11 » Fri Feb 08, 2008 10:22 pm

Unless I’m missing something, there has to be a problem with your vector DNA. It can’t be the enzymes—the controls are fine, if I understand you. The enzymes are not contaminated with nuclease or such—you still recover the pcr product after digestion, and since the single-digest controls appeared to work, there is no reason to suspect that the dd failed for the pcr product, so most likely they are fine. The gel per se seems to be OK and the purification kits work for the pcr product, I assume, so there’s nothing wrong with them either. Do you see chromosomal DNA or small mw RNAs in your vector DNA either before or after digestion and if you do, how much? You shouldn’t see any of those things if your vector DNA was made correctly and/or hasn’t been contaminated in the meantime. I’d be curious to see a picture of the gel with undigested and digested vector DNA.
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Re: problem with cloning/restriction digest

Postby ashlie23 » Sat Feb 09, 2008 5:02 pm

Dear blcr11, Thanks a lot for both replies. Yes. I too believe that the problem lies in the vector DNA to begin with. Yesterday, when I ran my uncut and dbl digested vector on a gel, the uncut was running at the same length as dbl digested. (my prof told me that it happens sometimes - it doesnt mean that uncut vector is bad.) I will be running the gel purified (digested) DNA on a gel today and will send you a gel photo - perhaps you would know more.

Please let me know once you look at the gel (obviously after I send it to you).
p.s. would adding the CIP buffer and enzyme prior to running on a gel for purification interfere with extraction?

Thanks a lot!

Here's my info.
blcr11 wrote:Unless I’m missing something, there has to be a problem with your vector DNA. It can’t be the enzymes—the controls are fine, if I understand you. The enzymes are not contaminated with nuclease or such—you still recover the pcr product after digestion, and since the single-digest controls appeared to work, there is no reason to suspect that the dd failed for the pcr product, so most likely they are fine. The gel per se seems to be OK and the purification kits work for the pcr product, I assume, so there’s nothing wrong with them either. Do you see chromosomal DNA or small mw RNAs in your vector DNA either before or after digestion and if you do, how much? You shouldn’t see any of those things if your vector DNA was made correctly and/or hasn’t been contaminated in the meantime. I’d be curious to see a picture of the gel with undigested and digested vector DNA.


----------------------------------------------------------------------------------------
you are seeing significant amounts of supercoiled and/or relaxed vector in your digests, then your enzyme(s) isn’t cutting very efficiently—or are you just worried about any residual uncut vector DNA? Is your vector DNA prep really old? Maybe you just need to make a fresh prep of vector DNA—or buy some more, though NEB seems to have discontinued the old IMPACT kit. I don’t know if the old vectors are still available. Or maybe you need to do the phenol extraction before you do the digestion on the assumption that an old prep has accumulated some kind of inhibitor. Another—remote—possibility is methylation. NdeI doesn’t care about methylation, but XmaI is inhibited by CpG methylation. But the methyl transferases responsible for CpG methylation are eukaryotic enzymes, not bacterial so far as I know. I guess this isn’t a very satisfying solution and it may not work (or you’ve tried it already), but if it were me, I would make or buy fresh pYTB2 vector, get fresh NdeI and XmaI, and do it again.
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