mysterious band after transformation and miniprep... - (Oct/19/2005 )

Hi, everyone,

I got a problem while i am selecting clones after transformation...

I did ligation between an insert(1.3Kb)and another one(6.0Kb).

The 1.3kb insert was got by cutting with XbaI and BgLII from one vector(2.46kb), and anther one(6.0Kb=vector + a part of cDNA) from another different vector(2.9kb) by the same cutting(XbaI and BgLII). Then,these two fragments will make a full-length cDNA after ligation.
I could see clearly both 1.3kb and 6.0kb right bands on the gel after fragments cleaning-up before the ligation. And the final band after ligation should be around1.3+6.0=7.3Kb if it's done successfully.

But, i screened 25 clones and repeated two times. I could get nothing but a mysterious band, which is around 3.8kb always. I don't know why? Anybody could give me some suggestions? Thanks.

Junda

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Just a guess Mysterious band 3.8Kb = vector A 2.46Kb + Insert A 1.3Kb
2.46 +1.3= 3.8Kb isn't it

Explanations:
Possibly some residual supercoiled plasmid A which give you transformants more effiently than your ligation !
Vector A is only cut on one side and religate which generates again same type of recombinants unfortunately not the one you're looking for !

Pesji

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I was thinking along the same lines as Pesji...

Explain again how you've done this: Is this final ligation a three-way ligation (1.3 kb fragment + 6.0 kb fragment + vector)?

Or does the 6.0 kb fragment constitute your vector, such that your cloning reation is 1.3 kb fragment + 6.0 kb vector (even if the vector contains some portion of your gene already)?

What is the selection here, and where does the marker reside?

Also, can you clarify the statement "I could get nothing but a mysterious band, which is around 3.8kb always"? Are you saying that when you digest the plasmid recovered from your transformants with Xba I and Bgl II it produces only a single band on the gel that runs at 3.8 kb, and no other bands at all?

Does the 1.3 kb fragment contain any carry-over vector sequences?

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Dear Pesji,

Thanks.
Sorry, for some clarified explanation first of all...

The final ligation is this: 1.3kb fragment +6.0kb(vector2.9 + a portion of gene already3.1Kb). Selection is ampicillin which is away from MCS (my 6.0vector is pbluescript(KS), another vector containing 1.3kb fragment is psp73 with a ampicilin marker as well).

I did the miniprep and cut the purified plasmid with enzymes one at a time(BgLII or HindIII, which have only one restriction site in consideration of whole construct(1.3kb+6.0kb). They all show almost the same size around 3.8kb. Only one cut(i just want to see the whole lenght of the construct) ... has nothing to do with XbaI+ BgLII cutting this time.

1.3kb fragment contains no carry-over vector sequence after the vector(2.9kb+4.3kbcDNA) was cut with XbaI +BgLII(6.0kb left after cutting). Before cutting, carry-over vector contains some portion of 1.3kb fragment from an0ther vector(psp73).

But i am still confused since i could see clearly 1.3kb band cut from 2.46vector on the gel and these two bands were seperated well, and i cut the seperated 1.3 band only, that's all. That means that residual supercoiled vector A was mixed with the 1.3 band i cut? Unbelievable. If it's the case, how to prevent this from happening?

Any possibility of contamination?

Best,

Junda

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[quote name='junda' date='Oct 19 2005, 03:46 PM' post='28325']
Dear Pesji,

Thanks.
Sorry, for some clarified explanation first of all...


But i am still confused since i could see clearly 1.3kb band cut from 2.46vector on the gel and these two bands were seperated well, and i cut the seperated 1.3 band only, that's all. That means that residual supercoiled vector A was mixed with the 1.3 band i cut? Unbelievable. If it's the case, how to prevent this from happening?

Any possibility of contamination?

Best,

Junda



Well as I already say in this forum in another topic Molecular Biology can be the most frustrating activity since you get sometimes very strange and hard to explain results believe me (20 years of practice).

Yes your insert can still be contaminated with a very small proportion of vector I know it's hard to explain cause I'm sure you took very much care to well separate the bands but it still can be trapped !

The problem is that SC DNA is much more efficient in transformations than any type of ligation !

Possible contamination source is incomplete cutting of the Vector A It's most of the time the explanation !

To come back to your contrôl digestion is HindIII in your insert or in the vector ?

I always checked my potential recombinant with an internal restriction site of the insert.

Another question already pointed out by Homebrew where you didn't answer I guess

Did you try to cut your minipreps with the XbaI and BglI ? Cause using one enzyme show you the size of the linear form of your complete A vector if you use both enzymes at the same time you might see your A insert again (1.3Kb) and the A vector (2.46Kb)

Pesji

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Dear Pesji,

Thank you very much.
HindIII is the only cutting site in the vector, and BgLII is only site in the 1.3insert. They were used seperately.
I did not cut my clones with XbaI and BgLII, maybe i should have done that.
By the way, any way to prevent such kind of contamination?

Best,

Junda
[quote name='pesji' date='Oct 19 2005, 07:09 AM' post='28327']
[quote name='junda' date='Oct 19 2005, 03:46 PM' post='28325']
Dear Pesji,

Thanks.
Sorry, for some clarified explanation first of all...


But i am still confused since i could see clearly 1.3kb band cut from 2.46vector on the gel and these two bands were seperated well, and i cut the seperated 1.3 band only, that's all. That means that residual supercoiled vector A was mixed with the 1.3 band i cut? Unbelievable. If it's the case, how to prevent this from happening?

Any possibility of contamination?

Best,

Junda



Well as I already say in this forum in another topic Molecular Biology can be the most frustrating activity since you get sometimes very strange and hard to explain results believe me (20 years of practice).

Yes your insert can still be contaminated with a very small proportion of vector I know it's hard to explain cause I'm sure you took very much care to well separate the bands but it still can be trapped !

The problem is that SC DNA is much more efficient in transformations than any type of ligation !

Possible contamination source is incomplete cutting of the Vector A It's most of the time the explanation !

To come back to your contrôl digestion is HindIII in your insert or in the vector ?

I always checked my potential recombinant with an internal restriction site of the insert.

Another question already pointed out by Homebrew where you didn't answer I guess

Did you try to cut your minipreps with the XbaI and BglI ? Cause using one enzyme show you the size of the linear form of your complete A vector if you use both enzymes at the same time you might see your A insert again (1.3Kb) and the A vector (2.46Kb)

Pesji
[/quote]

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Hi, there,

I cut the mysterious band with XBaI and BgLII, and the two bands after cutting basically match 1.3Kb insert and 2.46Kb vector. So, the whole thing was mixed with 1.3kb insert...

Then, I repeated two times of ligation, the ligation products i got still were not the one i want, which should be around 7.3Kb. One time is about 9Kb, another time ever larger than 9Kb. i usually picked up 10 clones for each ligation and transformation.

The right band is hard to get ever though the right size of ligation fragments shows up on the gel...

Any one can give me some suggestions and solutions? Thanks

Junda

[quote name='junda' date='Oct 19 2005, 06:57 PM' post='28426']
Dear Pesji,

Thank you very much.
HindIII is the only cutting site in the vector, and BgLII is only site in the 1.3insert. They were used seperately.
I did not cut my clones with XbaI and BgLII, maybe i should have done that.
By the way, any way to prevent such kind of contamination?

Best,

Junda
[quote name='pesji' date='Oct 19 2005, 07:09 AM' post='28327']
[quote name='junda' date='Oct 19 2005, 03:46 PM' post='28325']
Dear Pesji,

Thanks.
Sorry, for some clarified explanation first of all...


But i am still confused since i could see clearly 1.3kb band cut from 2.46vector on the gel and these two bands were seperated well, and i cut the seperated 1.3 band only, that's all. That means that residual supercoiled vector A was mixed with the 1.3 band i cut? Unbelievable. If it's the case, how to prevent this from happening?

Any possibility of contamination?

Best,

Junda



Well as I already say in this forum in another topic Molecular Biology can be the most frustrating activity since you get sometimes very strange and hard to explain results believe me (20 years of practice).

Yes your insert can still be contaminated with a very small proportion of vector I know it's hard to explain cause I'm sure you took very much care to well separate the bands but it still can be trapped !

The problem is that SC DNA is much more efficient in transformations than any type of ligation !

Possible contamination source is incomplete cutting of the Vector A It's most of the time the explanation !

To come back to your contrôl digestion is HindIII in your insert or in the vector ?

I always checked my potential recombinant with an internal restriction site of the insert.

Another question already pointed out by Homebrew where you didn't answer I guess

Did you try to cut your minipreps with the XbaI and BglI ? Cause using one enzyme show you the size of the linear form of your complete A vector if you use both enzymes at the same time you might see your A insert again (1.3Kb) and the A vector (2.46Kb)

Pesji
[/quote]
[/quote]

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Heu Junda sorry but it seems that you don't really know how to use the Quote option because now your post is so confuse that it's hard to understand what is new in your post . I paste what I thought to be your last answer !

So you have the explanation this mysterious band is the original vector that you carried along I also bet that the 9KB clones are the original vector B unfortunately and probably for the same reason of contamination !

If you repeat the ligation with the same material you will probably never be out of your trouble so just start from the beginning

1/ Cut vector A and make sure that it's 100% cut (at least 30 units of enzyme for 5micrograms of vector and 3 hours incubation in the correct buffer)

2/ Separate very well your products on an agarose gel I would rather use TAE buffer in your particular case

3/ Always do a religation control of the vector alone to be sure that you didn't carry along SC DNA.

Pesji

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Dear Pesji,

Thanks. a good method worth trying.

Jun

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Hi, guys,

It happened that i found a interesting method while i am surfing the internet.
The way i did is something like this basically: i treated the post-ligation mixture with a restriction enzyme at the site which is located only between two cutting sites of both vector A and B(XbaI and BgLII),
but make sure there is no such a cutting site in the insert(from vector A).
Therefore, there should be only expected recombinant constructs existed after the incubation with such a unique endonuclease, theoretically speaking, since all of other contaminating uncut vector would be got rid of transformation.

So, i got only 2 clones by doing this, and sequencing and gel running shows that they are the ones i want...

Thanks

Jun Wang

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