10 replies to this topic

[nipun]

Hi all
I transformed DH5-alpha with a plasmid vector carrying ampicillin marker gene. it showed growth on ampicillin plates. now when i tried to isolate plasmid from this transformed DH5Alphaa..  I got 4 bands in Agarose gel..
can anyone help me out to find what  exactly they are???
how can i isolate the plasmid of 9 kb i used to transform it...???

regards
nipun

[dpo]

did you digest your DNA before running the gel? if not, one plasmid can appear as different bands (see pic in attach)

Attached Thumbnails

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[nipun]

Thanks Dear
this may be a possibility..
Can you please tell me how to digest DNA before running Gel.?? I didnt run tris-acetate agarose gel..
I ran it on just 1% agarose gel, however the sample was stored in tris-acetate buffer.  
regards
nipun

Edited by nipun, 30 January 2009 - 03:12 AM.

[hanming86]

Name of the vector would help here.
Before you digest it would help to know what restriction sites are on the plasmid.
Then you would pick the appropriate restriction enzyme to cut the plasmid and then run on gel.

[nipun]

hanming86, on Jan 30 2009, 04:13 AM, said:

Name of the vector would help here.
Before you digest it would help to know what restriction sites are on the plasmid.
Then you would pick the appropriate restriction enzyme to cut the plasmid and then run on gel.

its pREP vector...
currently added to DH5Alpha from where i tried to isolate..
but after digesting with restriction enzyme how will i get a single band... will it be of linear plasmid opened from one site?? i guess so..

but the bands are too far from each other.. so is it possible to be of same plasmid in different folded forms give bands at large distances ??

regards
nipunh

[merlav]

If you didn't digest the plasmid what you are seen is the plasmid at different steps of coiling (the more coil the more it travel in the gel).  To isolate the plasmid  you can use a column base purification kit.  What you will do with the plasmid later???it will be easier to tell you what to do if I know the dowstream assay.

[perneseblue]

nipun, on Jan 30 2009, 04:25 AM, said:

but after digesting with restriction enzyme how will i get a single band... will it be of linear plasmid opened from one site?? i guess so..

Once you know what plasmid you have, down load its DNA sequence into plasmid management programme. You can then look at the sequence and find all the restriction sites for enzymes that your lab has. Pick one restriction enzyme that is cuts your vector. Depending on the number of restriction sites present in the vector for the restriction enzyme used, the vector can be cut to form a single linear DNA product or multiple DNA fragments.

Run out the digestion mix on a gel. If the number of size of the DNA fragments produce is what you expect (since you know the plasmid sequence), then it is very likely that your vector is the right thing. You can make several different restriction digest to confirm your plamsid.

nipun, on Jan 30 2009, 04:25 AM, said:

but the bands are too far from each other.. so is it possible to be of same plasmid in different folded forms give bands at large distances ??

Yes, the plasmid conformation has a strong effect on its movement through a gel. That is why you must cut/digest a sample of your vector to confirm that it has roughly the right structure.

[dpo]

nipun, on Jan 30 2009, 01:25 PM, said:

its pREP vector...
currently added to DH5Alpha from where i tried to isolate..
but after digesting with restriction enzyme how will i get a single band... will it be of linear plasmid opened from one site?? i guess so..

but the bands are too far from each other.. so is it possible to be of same plasmid in different folded forms give bands at large distances ??

regards
nipunh

I don't know this vector, and a quick google search didn't reveal any maps, but indeed, if the enzyme cuts only once, you will see one band of the linearized plasmid. Ideally, you select an enzyme that cuts 2 or 3 times so you get 2 or 3 bands.

The bands can be quite far from each other if they're all uncut, so I would definitely recommend digesting the plasmid. Maybe one of your colleagues has the sequence of this vector?

edit: scooped by perneseblue while looking for the map

Edited by dpo, 30 January 2009 - 03:56 AM.

[perneseblue]

I think I can help  

Unless there is another type of pREP plasmid which I am not aware off, the pREP vector is the generic name for a family of vectors used in S. pombe (Fission yeast aka the other yeast) which are structurally similar but differ in the strength of their nmt promoter.

So we have pREP1 (strongest), pREP3, pREP41, pREP81(weakest). You will have to determine which pREP vector you have, if worst comes to worst by sequencing.
Forsburg's website

Does anybody know of free plasmid management programme? I haven't been keeping track, and the list has gone. Vector NTI is 'no longer' free anymore.

(   I didn't mean to scope you dpo)

[nipun]

I don't know this vector, and a quick google search didn't reveal any maps, but indeed, if the enzyme cuts only once, you will see one band of the linearized plasmid. Ideally, you select an enzyme that cuts 2 or 3 times so you get 2 or 3 bands.

The bands can be quite far from each other if they're all uncut, so I would definitely recommend digesting the plasmid. Maybe one of your colleagues has the sequence of this vector?

edit: scooped by perneseblue while looking for the map
[/quote]

Thanks a lot dear..
I have the map and have information of cutting sites..
will digest it and confirm
regards
nipun

[nipun]

[quote name='perneseblue' date='Jan 30 2009, 05:15 AM' post='13901']
I think I can help  

Unless there is another type of pREP plasmid which I am not aware off, the pREP vector is the generic name for a family of vectors used in S. pombe (Fission yeast aka the other yeast) which are structurally similar but differ in the strength of their nmt promoter.

So we have pREP1 (strongest), pREP3, pREP41, pREP81(weakest). You will have to determine which pREP vector you have, if worst comes to worst by sequencing.
[url="http://www-rcf.usc.edu/~forsburg/vectors.html"]Forsburg's website[/url]


You are very right dear..
its pREP1 and i have its sequence, will follow your suggestions.
Thanks a lot for your help
regards
nipun