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mat agar dish and a cloning problem


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#1 Heini

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Posted 22 February 2005 - 02:43 AM

Hi there!

I have a question about a phenomenon in my ampisillin agar plate.

I've tried cloning 3,8kb PCR product to KpnI/BglII of the 7,5kb vector. This is a Kanamysin resistant vector. Despite several attemption I can't obtain any colonies after transformation into DH5a-cells. The kanamysin agar plates are clear. I have positive control (the intact vector) in transformation, which is ok. (I've asked this in topic cloning long fragments)

I've also tried another plasmid, which had KpnI/BglII sitse (pBLCAT2). I've digested it and gelpurified the bands 2,8kb and 1,7kb, and religated this. This is a ampisillin resistant vector. I can't get a single colony from this control ligation step either, but what I see in these ampisillin plates is a kind of mat surface. What is it?

Once, I also tried to religate HindIII cut a pcDNA3 plasmid and it gave me a same phenomenon. I took a swipe of this mat to new plate, but nothing grew there.

The only time i got some colonies is the time I didn't do gel purifying step to my fragments. (And I assume it all was backround, as I tested few colonies).

Any hints to my clonig problem are also welcome (I have tried different ligation temperatures, times, DNA amounts in transformation etc...)

Thank you in advance

Edited by Heini, 22 February 2005 - 05:39 AM.


#2 sharath

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Posted 26 February 2005 - 07:04 AM

First, you need to standardise you transfmation procedure with a standard plasmid. During transforation, the volume of the ligation mix should not exceed 1% of the volume of competent cells, and still that amount should contain around 10-100 ng of DNA.

For selection plats, use the antibiotics at concetration recommended for that particular vector.

In case of ampicillin selection marker, the transformants need to be incubated at 37C for 1hr in preheated LB broth (1ml) without ampicillin to allow the vector to express the ampicillinase enzyme.

Also, serially dilute your transformation mix before plating. The mat-growth is when you plate large amount of cells without dilution.
Sharath B.

#3 Heini

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Posted 01 March 2005 - 01:21 AM

Hi!
Thank you for your reply.
If I use the vectors used for cloning as a positive control, shouldn't it be enough control? As they both work in transformation and in plates (kanamysin and ampicillin respectively). I use 10-20 ng of these plasmids, and they give me a nice set of colonies on their plates. Shouldn't the procedure and antibiotic be then ok for those plasmids?

My transformation procedure is: I use 100Ál of cells, 0,5-20Ál ligation mix
Incbate on ice 30 min
heat shock 42C, 45-60s
immediately to ice, 2min
add 900Ál LB
+37 shaker 45-60 minutes
spin down 2000 rpm, 2min
take off 900Ál LB
resuspend cells
plate cells
inkubate +37 o/n

I think too much bacteria or DNA might be the reason to the mat. I'll try less both. Earlier I've used less DNA for transformations, but then I didn't even obtain a mat, and I still don't get anything in the kanamysin plates (even i've tried different amounts of DNA).
But as a CAT2 should work in theory, I'll try to dilute. Do you mean just take less transformation volume to plates or mean diluting all before plating?
(or to plate the whole 1 ml?)

Thanks

#4 Heini

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Posted 04 March 2005 - 12:28 AM

Hi!
I now used 1,5Ál (~11ng) of ligation mix to 150Ál of cells. I plated 1/10 of the cells I've used to, but the mat plate still seems to appear.
I'm quite confused about this.

Heini :(

#5 siri

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Posted 04 March 2005 - 08:15 PM

Hi!
Thank you for your reply.
If I use the vectors used for cloning as a positive control, shouldn't it be enough control? As they both work in transformation and in plates (kanamysin and ampicillin respectively). I use 10-20 ng of these plasmids, and they give me a nice set of colonies on their plates. Shouldn't the procedure and antibiotic be then ok for those plasmids?

My transformation procedure is: I use 100Ál of cells, 0,5-20Ál ligation mix
Incbate on ice 30 min
heat shock 42C, 45-60s
immediately to ice, 2min
add 900Ál LB
+37 shaker 45-60 minutes
spin down 2000 rpm, 2min
take off 900Ál LB
resuspend cells
plate cells
inkubate +37 o/n

I think too much bacteria or DNA might be the reason to the mat. I'll try less both. Earlier I've used less DNA for transformations, but then I didn't even obtain a mat, and I still don't get anything in the kanamysin plates (even i've tried different amounts of DNA).
But as a CAT2 should work in theory, I'll try to dilute. Do you mean just take less transformation volume to plates or mean diluting all before plating?
(or to plate the whole 1 ml?)

Thanks

just try with some other electrocompetent cells. I use the same protocol the one which you used. Some time competent cells may loose effeciency thats why you dont get any colonies
-siri




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