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Ligation problem

ligation

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#1 huyenpham

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Posted 31 August 2011 - 04:59 PM

Hi guys,
I have to clone a 600bp-insert to a 7kb-plasmid by using sequential digestion AsisI and MluI. I sequentially cut my vector and PCR product of my insert and ligated with different ratio of insert: vector (3:1, 5:1, 10:1) by using Ligation mix from Takara at 16oC/4hrs or 16oC overnight. It happened 2 months ago when I had so many colonies but no insert inside. Suddenly one day I got no colony at all even I used the same vector, insert, reagents and protocol. And up to now, I am still in no colony situation. I don't understand why reaction changes so dramatically.
But it was not the craziest thing. To check, I transformed the competence cell with the intact vector --> I got so many colonies --> vector is good and competence cell as well as plate and transformation procedure is OK.
Then , I took the intact plasmid --> ran 2 microgram on gel. I observed as usual: 1 supercoiled band at 6k , 1 nicked band at > 10kb and 1 band at the expected size 7kb that smeared together with the 6kb band. I cut the 7 and 6 kb band and purify. After purification, I ran 1 microlitre on gel and I also saw 3 band --> was it strange??? Because I cut only the 6 and 7kb band??? Then I transformed the purified product and I got no colony at all. I waited for more than 24 hrs but I even did not have any satellites or opportunistic colony.
I repeated it several times and always had the same result.
What it the problem?? any suggestion???

#2 ranvi

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Posted 31 August 2011 - 06:31 PM

I know this it was bothering me for a mo just got it figured out. sub cloning driving me crazy all sorts of weird results.
Heres some sloution you can try
1. try the known plasmid with the same vector, just do the transforamtion
2. the cut some of the same plasmide with the enzyme you want to cut , then do the pcr clean up , do the ligation and transformation
3. cut the same plasmid and gel purify that then do the ligation and transformation
4. compare the results
I did all of those I got colonies on the uncut and cut and pcr cleaned but not on gel purified
i was using 1.5- 2% gel instead of 0.7 -0.8 % which was inhibiting the ligation also there is dna clean up kit by zymo research its called dna clean up and concentrator it works very well use that

Then I did cut the vector and run on 0.7% gel and I got positive colony

good luck

Edited by ranvi, 31 August 2011 - 06:31 PM.


#3 perneseblue

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Posted 31 August 2011 - 07:32 PM

I can't quite work out what is happening from your post. Could you write step by step what was done.As you are well aware, it is the details that get you.

As this is a ligation between a vector and PCR product,

The first question would be, how many bp are there 5' to the restriction site? I would use at least 6bp. Too few bp and the enzyme can not bind to the restriction site and cleave it efficiency. Example : according to NEB, 2bp overhang for MluI only results to 50% after 20hr of digest.

The next question is how did you purify the PCR product? Column? Phenol? (Archean DNA polymerase are resistant to phenol, you no inactivation there.)

Did you gel purify the PCR product?

How did was the DNA cut? How long, what was the digest formula? In what buffer?

According to NEB, both AsiSI and MluI can work in NEB buffer 3. You do not need to do a sequential digest.

Did you just digest the vector with AsiSI and MluI, and then transformed the linear DNA into cells? (Am I reading this correctly... very odd thing to do)
If done correctly, no colonies should appear. Linear DNA will not propagate. And AsiSI and MluI do not form compatible ends. No ligation is possible.

If I am reading your post correctly, the multibanded pattern seen is actually due to incomplete digestion of the vector. Ie the vector is not completely cut. You should digest longer.

Please supply more details of what exactly was done.
May your PCR products be long, your protocols short and your boss on holiday

#4 huyenpham

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Posted 31 August 2011 - 08:39 PM

I can't quite work out what is happening from your post. Could you write step by step what was done.As you are well aware, it is the details that get you.

As this is a ligation between a vector and PCR product,

The first question would be, how many bp are there 5' to the restriction site? I would use at least 6bp. Too few bp and the enzyme can not bind to the restriction site and cleave it efficiency. Example : according to NEB, 2bp overhang for MluI only results to 50% after 20hr of digest.

The next question is how did you purify the PCR product? Column? Phenol? (Archean DNA polymerase are resistant to phenol, you no inactivation there.)

Did you gel purify the PCR product?

How did was the DNA cut? How long, what was the digest formula? In what buffer?

According to NEB, both AsiSI and MluI can work in NEB buffer 3. You do not need to do a sequential digest.

Did you just digest the vector with AsiSI and MluI, and then transformed the linear DNA into cells? (Am I reading this correctly... very odd thing to do)
If done correctly, no colonies should appear. Linear DNA will not propagate. And AsiSI and MluI do not form compatible ends. No ligation is possible.

If I am reading your post correctly, the multibanded pattern seen is actually due to incomplete digestion of the vector. Ie the vector is not completely cut. You should digest longer.

Please supply more details of what exactly was done.

Hi perneseblue,
1. Like you, I used 6bp to the 5' restriction site.
2. How I purify my PCR product? I tried so many ways: run on gel --> purify, purify w/o gel, ethanol precipitation. all gives me no subcloning. By the way, problem í 2 months ago, I got so many colonies with no insert. But now, I have no colony at all.
3. I think u misunderstand what I wrote. To check what step can make trouble, I decided to make control. My first control is transformation with vector --> have so many colonies. My 2nd control is transform the vector (un cut, just add 2microgram vector to 50 microlitre water and then run on gel, then cut the gel and purify) --> no colony. The multi band that u concerned appeared when I ran the uncut vector, and this is the normal pattern of a plasmid.
The question is that why I did not do any thing to my vector, only run on gel and purify, but I got no colony at all.

#5 huyenpham

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Posted 01 September 2011 - 10:11 PM

I know this it was bothering me for a mo just got it figured out. sub cloning driving me crazy all sorts of weird results.
Heres some sloution you can try
1. try the known plasmid with the same vector, just do the transforamtion
2. the cut some of the same plasmide with the enzyme you want to cut , then do the pcr clean up , do the ligation and transformation
3. cut the same plasmid and gel purify that then do the ligation and transformation
4. compare the results
I did all of those I got colonies on the uncut and cut and pcr cleaned but not on gel purified
i was using 1.5- 2% gel instead of 0.7 -0.8 % which was inhibiting the ligation also there is dna clean up kit by zymo research its called dna clean up and concentrator it works very well use that

Then I did cut the vector and run on 0.7% gel and I got positive colony

good luck

I tried 0.7% gel yesterday but it did not give me any colony.

#6 ranvi

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Posted 02 September 2011 - 03:46 AM

did u do the pcr clean up?

which gel clean up kit you are using or using exposing the gel uv for a long time? it got to be less than 20 sec ask some one to watch u while u are performing
usually qiagen is better for gel extraction when you cut the pieces purify only 2 well per column
overloading might be a problem add iso propanol in gel extraction too

#7 prodes

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Posted 02 September 2011 - 12:13 PM

Something more dramatic is going wrong for you which has most probably nothing to do with the gel percentage, gel extraction kit or PCR clean up kit.

If your transformation and cells are alright your problem most probably lies in the quality of your vector and/or insert. One possibility is that the vector/insert is degraded due to star activity or contamination of your solutions. To check that 1) Run uncut and cut plasmid (if there is star activity or contamination, most probably you will see multiple bands of the cut vector or smearing) 2) cut the target plasmid with a single restriction enzyme at a time and religate it - the product should give colonies and is a positive control for the quality of your enzymes and contamination of your solutions.

Did you start dephosphorylating your vector when you stopped seeing colonies? The fact is that this cloning didn't work if you didn't get inserts even when you had colonies. So, the problem is not the colony loss per se.

Keep positive and negative controls from start till the end: for enzymes, for ligation, for transformation.

Edited by prodes, 02 September 2011 - 12:13 PM.


#8 perneseblue

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Posted 06 September 2011 - 04:59 PM

Okay, I think I understand what is going on.
There is uncut DNA being gel purified and re-transformed into cells.

Given that direct transformation of DNA to cell works, it means the cells are competent and electroporator works.

That means, the DNA is being lost somewhere. After gel purification of the DNA, you should go run a sample on a gel to verify that you do have DNA. Is the DNA being exposed to UV light for very long period of time?
May your PCR products be long, your protocols short and your boss on holiday





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