8 replies to this topic

[Ramare]

Dear Gurus,

Do you all have any suggestions on how to separate two plasmids ( size 11761 bp and ~12000 bp) using agarose gel electrophoresis, I had consider using pulsed field gel electrophoresis, but I did not know whether it will work or not?



It is because recently I had to construct plasmid where the initial vector size is 11761 bp, then by using restriction endonucleases then ligation, the final product will be ~12000 bp, so, the size difference between vector and product is ~300 bp.

Then after ligation and transformation on agar plate, the recombinant plasmid agar shows more colonies than the negative control ( 1 site digested vector), but after I amplify the colony and extract their plasmid, it was all false results (I use HIT-JM 109 competent cell for transformation). These results had been repeated for 10 times, and this construct is important for my project.

Thats why I try to separate the ligation product (but do not know how) then transform it into the competent cell

Below is the procedure I do for the cloning process,

1. Digest Vector with NotI RE (1 site) and CIP for 3 hours
2. Amplify insert using PCR then after gel electrophoresis and purification, digested using NotI RE (2 sites) for 12 hours <-- I was desperate, then purify.

3. Quick ligate the vector and insert (ligase : Promega) in 20-22 celcius for 1 hour, V:I = 1:3
4. Transform into competent cell using 150 ng ligation product.
5. Pour it into agar plate with ampicilin and incubate 37 celcius for 12-16 hours.
6. Isolate and amplify colonies.

Did you find any quirks on my methods or things that should be change, suggestions is greatly welcome. Because this is my first time constructing plasmid.

Thanks!  

[swanny]

Ramare, on Apr 27 2009, 01:57 PM, said:

Dear Gurus,

Do you all have any suggestions on how to separate two plasmids ( size 11761 bp and ~12000 bp) using agarose gel electrophoresis, I had consider using pulsed field gel electrophoresis, but I did not know whether it will work or not?



It is because recently I had to construct plasmid where the initial vector size is 11761 bp, then by using restriction endonucleases then ligation, the final product will be ~12000 bp, so, the size difference between vector and product is ~300 bp.

Then after ligation and transformation on agar plate, the recombinant plasmid agar shows more colonies than the negative control ( 1 site digested vector), but after I amplify the colony and extract their plasmid, it was all false results (I use HIT-JM 109 competent cell for transformation). These results had been repeated for 10 times, and this construct is important for my project.

Thats why I try to separate the ligation product (but do not know how) then transform it into the competent cell

Below is the procedure I do for the cloning process,

1. Digest Vector with NotI RE (1 site) and CIP for 3 hours
2. Amplify insert using PCR then after gel electrophoresis and purification, digested using NotI RE (2 sites) for 12 hours <-- I was desperate, then purify.

3. Quick ligate the vector and insert (ligase : Promega) in 20-22 celcius for 1 hour, V:I = 1:3
4. Transform into competent cell using 150 ng ligation product.
5. Pour it into agar plate with ampicilin and incubate 37 celcius for 12-16 hours.
6. Isolate and amplify colonies.

Did you find any quirks on my methods or things that should be change, suggestions is greatly welcome. Because this is my first time constructing plasmid.

Thanks!  

Who gave you the protocol? perhaps they can give you some hands-on advice.

In the meantime...
I don't usually use CIP (I prefer Antarctic phosphatase, as it's easier to kill),  but I would have thought 2 hours is too long. Check the product insert to see the recommended reaction conditions. Also, do you run the vector out on a  gel to check for complete digestion, and gel- purify?

You can test your insert digestion, as well as the ligase itself, by this quick test. Once you have killed the RE (!!!), treat some of the insert with ligase for ~20 minutes at RT; also, do the same to some DNA size markers. Run samples out on a gel. Both should make ladders, and the DNA marker bands should move up the gel.

When doing your ligation, don't just do the quick one. Try an overnight as well, and transform this the next day.

[perneseblue]

Ramare, on Apr 26 2009, 07:57 PM, said:

Do you all have any suggestions on how to separate two plasmids ( size 11761 bp and ~12000 bp) using agarose gel electrophoresis, I had consider using pulsed field gel electrophoresis, but I did not know whether it will work or not?

Unfortunately it is not possible to separate a 11.7kb plasmid from a 12kb plasmid. PFGE will not help either. Separation of plasmids between 10-20kb in size is best done on a regular gel rather than a PFG.

Given the problem that you are facing it would be better to conduct restriction digest mapping or colony PCR to screen for your plasmid.

As noted by Swanny, 3hrs of CIP is too long. This enzyme will damage the ends of your vector rendering it unligatable. Do not over dephosphorylate with CIP. A different enzyme such as SIP would be better if you want to digest and dephosphorylate your vector at the same time.

Also note that NotI restriction site requires a minimum of 8bp skirting each end of the restriction site before it can be cleaved by the NotI enzyme efficiently.  Where enough bp added to the NotI site on the PCR product?

[little mouse]

instead to screen by looking at the size of the whole plasmid, I would digest it to excise the insert and screen by looking at the size of the insert.

[Ramare]

To Swanny

Hey Swanny,

First of all thanks for the suggestions, I had done your what you are suggesting by doing the o/n ligation in 16 celcius, I hope it works (finger crossed).

And yes, after the vector NotI digestion, I run gel electrophoresis then gel purify.

The reason I apply 3 hours digestion of vector using NotI and CIP is because. Our lab usually put 2 hours for NotI alone, and because now I also put CIP, so, I think I should extend the time, because NotI will cut the plasmid first, then dephosporylated by CIP. If this time it still does not work, I will reduce the vector digestion time.

Thank you ^^


To Perneseblue

Good day Perneseblue!

Thanks for the insight you gave me.

I put 3 bp on the PCR product, where according to the enzyme protocol will produce around 60-80% digestion efficiency. Next time I will reduce my vector digestion time, and I will ask my supervisor whether or not we will buy SIP, is it expensive? ^^

Thank you very much for the answer! It helps alot, mostly the digestion part.


To little mouse

Halo Little mouse,

Thanks for the answer, but....

Actually I don't really get what you are trying to say. But if you mean that I should excise the plasmid after ligation to see if it had been ligated, then it was the problem, as I do not know how to separate the vector and vector + insert plasmid.

Or if what you are meaning that after transforming --> amplify growing colony --> extract plasmid --> the excise using RE to see whether the insert is there, then, I had done around 100 amplification and most of the plasmid sizes is > 10kb.

So, basically my idea is after ligation I would like to separate the succesful ligation product from the unligated vectors, then gel purify the ligation product, then using the purification product to transform the competent cell.

Thank you ^^

Edited by Ramare, 27 April 2009 - 10:24 AM.

[swanny]

Ramare, on Apr 28 2009, 03:57 AM, said:

To Swanny

Hey Swanny,

First of all thanks for the suggestions, I had done your what you are suggesting by doing the o/n ligation in 16 celcius, I hope it works (finger crossed).

And yes, after the vector NotI digestion, I run gel electrophoresis then gel purify.

The reason I apply 3 hours digestion of vector using NotI and CIP is because. Our lab usually put 2 hours for NotI alone, and because now I also put CIP, so, I think I should extend the time, because NotI will cut the plasmid first, then dephosporylated by CIP. If this time it still does not work, I will reduce the vector digestion time.

Thank you ^^


To Perneseblue

Good day Perneseblue!

Thanks for the insight you gave me.

I put 3 bp on the PCR product, where according to the enzyme protocol will produce around 60-80% digestion efficiency. Next time I will reduce my vector digestion time, and I will ask my supervisor whether or not we will buy SIP, is it expensive? ^^

Thank you very much for the answer! It helps alot, mostly the digestion part.


To little mouse

Halo Little mouse,

Thanks for the answer, but....

Actually I don't really get what you are trying to say. But if you mean that I should excise the plasmid after ligation to see if it had been ligated, then it was the problem, as I do not know how to separate the vector and vector + insert plasmid.

Or if what you are meaning that after transforming --> amplify growing colony --> extract plasmid --> the excise using RE to see whether the insert is there, then, I had done around 100 amplification and most of the plasmid sizes is > 10kb.

So, basically my idea is after ligation I would like to separate the succesful ligation product from the unligated vectors, then gel purify the ligation product, then using the purification product to transform the competent cell.

Thank you ^^

Hi,
Your NotI digestion should be complete in an hour, assuming your DNA is of high quality: if you're worried, use 5 units per ug DNA. One of the guys in our lab recently learned the hard way that DNA quality is absolutely vital to good digestion, and that can come down to what cell strain was used to grow the vector (for example, NotI is sensitive to CpG methylation), as well as which media was used (LB seems to give lower cell yield, but better quality DNA, than 2YT).
For CIP treatment, the NEB website recommends 30 minutes @37C, so your incubation time is far too long. Try 30 minutes and see what you get. Don't forget to kill the enzyme after the reaction, or it will start dephosphorylating your insert during the ligation step!
pernes' point is your PCR product might not have enough bases at each end to allow good NotI cutting: best bet would be to order new primers with at leaast 8 bp before the NotI site.
Mouse's point is to do minipreps of your transformants and digest with NotI. If a clone has the insert, you will cut it out and will see it on a gel, along with linearised vector. Religated vector (that is, no insert) will simply be linearised.

Keep going, your problems will resolve themselves!

[HomeBrew]

I agree with the others, especially that your CIP step is too long.  Try adding the CIP to your restriction digest during the last 15 - 30 minutes (I do 5 minutes using 1/10th the recommended amount, and it works fine for me).  Are you gel purifying your vector after it's been digested and CIP treated?  If not, I would highly recommend that you do that; just run it on the same gel as your PCR product.

[little mouse]

Sorry, I answered too fast, but Swanny got the point.
Do a miniprep, digest with one or two enzymes that have site flanking the gene you inserted, or with one site inside the gene would even be better (together with an other site) so that you will obtain two bands instead of only one.
.... I'm desperately not clear.

you have two options.
the best would be to have a unique restriction site in the gene you inserted, and not in the previous vector where you inserted a gene.
you would digest with this enzyme and a second one.
if the gene is inserted, you will obtain two bands. if the gene is not inserted, you will only obtain one band.

If you don't have a unique restriction site in the gene, you can cut with the enzyme you used to subclone.
if you did or not insert the gene, in both case you will have two bands, but the smallest band will not have the same size (the bigger neither, but you won't be able to see the difference).

I hope my explanation are better now !

[Ramare]

Hello everyone!

Thanks for all the reply!

I had change my procedure and shorten the CIP time, hope it will works.

祝研安順利!

(means "Hope that all your research safe and smoothly going)

From Taiwan,
Ronald