6 replies to this topic

[light_my_way]

Hi,

I did an agarose gel run for my plasmid, pGEX-4T. I was surprised to see bands appearing at two positions. One was at more than 10000 bp and the other was at around 5000bp. The latter is close to the molecular size of pGEX-4T. So I am wondering what could the other band be? Also, there were fluorescent in the well, indicating that some plasmid DNA didn't move. Why is it so?

[george@CASE]

did you purify your plasmid from bacteria?

You may have genomic DNA (bacterial) contaminating your plasmid.

[Simonsays]

Is your DNA (pGEX) a product of a PCR? If so did you purify it on gel... it sounds like you have some template (pcDNA3 or another big vector) from which you performed a PCR.
It's only a hint... (it actually happened to me a while ago!!!)

Simon

[light_my_way]

Hi,

Thanks for the replies.  

The pGEX is not a product of PCR. It was however used for miniprep after transforming it into E.coli. So does it mean that it is the genomic DNA of E.coli that constitutes the band?  

Edited by light_my_way, 15 February 2005 - 03:20 AM.

[marc_U7snurp]

light_my_way, on Feb 15 2005, 04:19 AM, said:

Hi,

Thanks for the replies. 

The pGEX is not a product of PCR. It was however used for miniprep after transforming it into E.coli. So does it mean that it is the genomic DNA of E.coli that constitutes the band? 

Hiya,

did you digest your plasmid, before you let it run on the gel? If not you will have three bands:

The lowest are the supercoiled plasmids, then the more relaxed plasmids and in the end some concatamers (if I remeber this is the name... ;-))

Hope this might help

[Beta Campos]

I have a hard problem with pGEX4T1... without solution  at moment...
I make to especific primers for polylinker of pGEX4T1 with clivage BamHI and SmaI and my insert with 2600 bp clivage with the same enzymes.
I dont have a clone with insert...
Just a product in agarose gel with 10kb.
My boss think that are 2 empty vector clonage....
Help me... I tried many shots...
Tks.

Beta.

[swanny]

Beta Campos, on Mar 16 2009, 08:20 AM, said:

I have a hard problem with pGEX4T1... without solution  at moment...
I make to especific primers for polylinker of pGEX4T1 with clivage BamHI and SmaI and my insert with 2600 bp clivage with the same enzymes.
I dont have a clone with insert...
Just a product in agarose gel with 10kb.
My boss think that are 2 empty vector clonage....
Help me... I tried many shots...
Tks.

Beta.

Digest your plasmid with BamHI and SmaI. If your product comes back to plasmid monomer, then your boss is right. If you get plasmid plus something else, then you have cloned. Assuming your size data is correct, I think your boss is probably correct. How much vector and insert did you use in your ligation reaction, and what was the volume of the reaction?