11 replies to this topic

[myself]

Hi everybody,

Recently I was cloning an 0.7kb insert that is a part of 3'UTR region into a 7kb vector. At the end of my work I got colonies which turned out to all have a self ligated plasmid(I checked it with a PCR after doing minipreps using primers on the both side of the restriction site). That is what I did briefly:

1.I planned to do a single restriction site cloning and later to identify the colonies with the right orientation of the insert using PCR.
2.I cut the vector with HindIII enzyme in the Fermentas R buffer and added in the end 1ul of CIP and left it for 45min in 37C. I did a PCR clean up and checked my vector on electrophoresis and was linerized and all right. I left it in -20C, because I had to delay the rest of the experiment for two days.
3.I did a PCR for my insert using primers with the HindIII site on both sides with excessive 2 base pairs to ensure the activity of enzyme.
4. I digested the insert and cleaned it also with PCR cleanup, and checked the product on Agarose gel and it was also alright.
5. After measuring the densities and carried out a ligation using NEB quick ligation kit according to the protocol.
6. I transformed the bacteria, got colonies and checked them first with colony PCR but after getting no result I checked it with after doing minipreps. So I got no band for a product designed to be at the junction of the insert and vector but I got bands indicating that the vector selfligated.
I had two inserts. One of them gave something  about 20 colonies so I picked 9 of them, and the other one 7 colonies , so I picked all of them for the cloning. As you see the number of colonies also wasn't that satisfactory.

The thing is that both of my CIP and Ligase are expired but not too much(about one year in good storing conditions without thawing) so I don't think this is the problem. But to make it clear, can I assume that my ligase worked properly since I have a self ligated product? Or it can by that my CIP didn't work properly and anyway it got self ligated vector regard less of the ligase?
Are there any other options for this experiment to go wrong except for the bad action of these two enzymes?

Thank you guys for all your advices in advance.

[myself]

Another question is, can I for example set up differnt kinds of ligation reactions and check them with PCR before transfomation to see if it worked and spare one culturing and miniprep step?

[phage434]

It's very common to see high background of the kind you have when doing single enzyme cuts.  Any plasmid which is uncut by the HindIII digestion will transform very effectively, and it is very difficult to guarantee full digestion.

You can do several things: digest with two enzymes (suggested in any case to get directionality of the cloning).  Even then, you will have background.  Another approach is to PCR amplify the vector.  This allows you to use very small amounts (< 1 ng) of vector template, which is a good start at avoiding background.  Then, you can cut the PCR product with DpnI, to cut the vector template (but not the PCR product). Then, purify the PCR product, cut with the two enzymes you have created cloning sites for at each end, mix with your similarly prepared insert, ligate, and transform.

You probably should be using normal ligase, not quick ligase, which is best left for blunt end only ligations.

I hate CIP, it has never helped and often hurt my cloning.

Your colony pcr probably failed due to too many cells in the PCR.  Dilute a small amount of colony into 50 ul of water, then use 1 ul of this as the pcr template.  Do an initial cycle of 96-98 C for 5 minutes, then normal cycling.

[myself]

Thanks a lot for your quick answer.

[bsksln]

myself, on 19 December 2010 - 04:19 PM, said:

Hi everybody,

Recently I was cloning an 0.7kb insert that is a part of 3'UTR region into a 7kb vector. At the end of my work I got colonies which turned out to all have a self ligated plasmid(I checked it with a PCR after doing minipreps using primers on the both side of the restriction site). That is what I did briefly:

1.I planned to do a single restriction site cloning and later to identify the colonies with the right orientation of the insert using PCR.
2.I cut the vector with HindIII enzyme in the Fermentas R buffer and added in the end 1ul of CIP and left it for 45min in 37C. I did a PCR clean up and checked my vector on electrophoresis and was linerized and all right. I left it in -20C, because I had to delay the rest of the experiment for two days.
3.I did a PCR for my insert using primers with the HindIII site on both sides with excessive 2 base pairs to ensure the activity of enzyme.
4. I digested the insert and cleaned it also with PCR cleanup, and checked the product on Agarose gel and it was also alright.
5. After measuring the densities and carried out a ligation using NEB quick ligation kit according to the protocol.
6. I transformed the bacteria, got colonies and checked them first with colony PCR but after getting no result I checked it with after doing minipreps. So I got no band for a product designed to be at the junction of the insert and vector but I got bands indicating that the vector selfligated.
I had two inserts. One of them gave something  about 20 colonies so I picked 9 of them, and the other one 7 colonies , so I picked all of them for the cloning. As you see the number of colonies also wasn't that satisfactory.

The thing is that both of my CIP and Ligase are expired but not too much(about one year in good storing conditions without thawing) so I don't think this is the problem. But to make it clear, can I assume that my ligase worked properly since I have a self ligated product? Or it can by that my CIP didn't work properly and anyway it got self ligated vector regard less of the ligase?
Are there any other options for this experiment to go wrong except for the bad action of these two enzymes?

Thank you guys for all your advices in advance.

Well, brother, i was recently struggling with this problem too. now I am sick of picking colonies and I hate single digestion!
after struggling for 2 weeks, a friend of mine suggest me to use in fusion system from clonetech. they use this system to do molecular cloning now. this system is very fast, and does not need restriction enzyme and ligase. it is not expansive. my friend suggested me to use a half volume of suggested. tonight I will get the result, so exciting.
I think you can consider this method.

[myself]

Let us know how it worked.

Cheers!

[bob1]

If the vector is a problem - dephosphorylate it!

[Adrian K]

bsksln, on 20 December 2010 - 01:32 AM, said:

Well, brother, i was recently struggling with this problem too. now I am sick of picking colonies and I hate single digestion!
after struggling for 2 weeks, a friend of mine suggest me to use in fusion system from clonetech. they use this system to do molecular cloning now. this system is very fast, and does not need restriction enzyme and ligase. it is not expansive. my friend suggested me to use a half volume of suggested. tonight I will get the result, so exciting.
I think you can consider this method.

I had tried in-fusion... however it doesn't work for my case and is quite expensive...
How do you find it?

[bsksln]

adrian kohsf, on 22 December 2010 - 09:34 AM, said:

bsksln, on 20 December 2010 - 01:32 AM, said:

Well, brother, i was recently struggling with this problem too. now I am sick of picking colonies and I hate single digestion!
after struggling for 2 weeks, a friend of mine suggest me to use in fusion system from clonetech. they use this system to do molecular cloning now. this system is very fast, and does not need restriction enzyme and ligase. it is not expansive. my friend suggested me to use a half volume of suggested. tonight I will get the result, so exciting.
I think you can consider this method.

I had tried in-fusion... however it doesn't work for my case and is quite expensive...
How do you find it?

I have successfully constructed my plasmid using infusion. after 2 weeks struggling with single digestion, my friend suggested me to use in fusion system. the next day I got 12 positive clones(100%).
considering the price of restriction enzyme, ligase, gel extraction kit, and my time, I think I would choose in fusion.

[bsksln]

ps. my friends suggest me to use 1/4 or 1/2 volume when using in fusion. it works too. so the cost decreases.

[Adrian K]

hi thanks... i will troubleshoot my work again and see how it goes...

[genesquirt]

bob1, on 20 December 2010 - 01:27 PM, said:

If the vector is a problem - dephosphorylate it!

I agree with this, this is an easy step. I always dephosphorylate my vector before ligation.