Hey,
I have clonned a double stranded oligo into glo-mir vector.
My vector is 7.5kb and insertion is 81bp.
I have isolated my plasmid from colonies after trsnsformation and perform agarose gel electrophoresis %1 agarose with tae.
Interestingly my isolated plasmids gave bands upper than control load( not inserted empty vector)
I have overload my empty vector can this cause that problem?
I Will digest my vector with restriction enzymes than check for 81bp frsgment with na-borate gel. But I Wonder that, this bands can be a little up because of high load of empty vector or they are wrong bands.
1kb marker, my plasmids from different colonies, and the Last bandında is empty vector
Hai Enes, for 81bp product you, have to run the gel2% with 50bp ladder and 1kb ladder at opposite side also. Moreover 81bp is very small product you may need heigh concentration of plasmid for RE since the detection of DNA under gel doc need minimum of 20 to 100ng of DNA. To make at least 50ng, imagine how much Released insert needed. You may use online calculator to measure the amount of plasmid needed to meet 50ng insert.
Hi Enes, also you need to run your insert 81bp and empty vector as markers of your digestion. In my opinion the suitable agarose will be 1.5% and let the run for long time until u obtain a good separation
Good luck
I agree with both of the above but would only run the gel for a very short time since you are not really interested in the large bands but the very small band will be weak and will diffuse the further it runs so do not run it very far and accept that the larger bands will not be separated from each other, Run the gel at low voltage to minimise thermal diffusion of the band which will be sharper in a cooler gel
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