9 replies to this topic

[neoplazi]

Hi Dear Researcher,

I have a problem with transformation. Actually I do not know where is the problem. I am trying maybe 80th times but I am not finding true bands view .

I want to tell whole process.

I used head shock method. 100 ul E.Coli DH5α and 2 ul plasmid DNA (pCDF1-JAK2-GFP  10241 bp)

30 min. ice--> 1 min. 42 °C --> 2 min. ice --> 900 ul SOC media --> 1 hour 225 rpm 37 °C shaker -->Spreading to Agar Plate (fresh ampicilin also I added extra ampicilin)

12-14 hours inc.

I am getting a lot of colony.

Seeding to 5 ml LB + amp --> 8 hours 225 rpm 37 °C shaker --> Miniprep (used QIAgen minikit)

I am gettin good amount of DNA

There is no problem so for.

After enzyme digestion ( I tried 3-4 different enzyme 7-8 different configuration) I am facing incorrect bands and result.

I really need your suggestions. Please help me about this problem

*I checked LB, LB agar, ampicilin amount, temperatures, E. Coli efficiency, enzyme efficiency, used uncut control and positive control.

[phage434]

What evidence do you have that the original plasmid is correct?

[neoplazi]

I have the plasmid map.

So I am checking bands from it.

[phage434]

I would suggest that you have strong evidence that the sequence of the plasmid you have started with is incorrect.

[neoplazi]

But the plasmid is not new!

We took from different group that has been worked long time.

So there is no problem with construction of plasmid.

[bob1]

I agree with Phage - either you have the wrong plasmid (mistakes happen, mutations happen) or the map is incorrect.  Sequence the necessary bits of the plasmid to confirm properly.

[phage434]

After many years working in the lab and finding this sort of problem, I now first routinely sequence any plasmid coming into my lab from somewhere else. Trust, but verify.

[neoplazi]

Dear Phage and Bob,

Thanks for your interest.
You are kind and helpful.

After your posts I digested main (stock) plasmid with other transformed colony miniprep results.
Here my gel image:

I was finding extra and incorrect band (end of hole) and I saw it in stock plasmid.
Do I have plasmid contamination?

I do not have sequence posibility for now.
Can you offer another suggestion?

Attached Thumbnails

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Edited by neoplazi, 13 February 2013 - 06:58 AM.

[phage434]

Your gel did not show up. I would suggest that if you cannot sequence the plasmid, you should consider whether the plasmid is sufficiently close to what you want to be functional. Often minor modifications will not affect results. Perhaps your experiments can go on with the plasmid you actually have. It is unlikely to be contamination, but rather a mutation (or incorrect sequencing previously) since you get the same results following your transformation and miniprep.

[neoplazi]

After your suggestion also I want to sequence it. But unfortunately my supervieser has stuck mentality and forcing me for extra repeat.

Good bless me

Edited by neoplazi, 15 February 2013 - 09:14 AM.