I performed a ligation of 1.5 kb insert into a 4.2 kb vector and I got colonies on my kanamycin plate. I picked up 8 colonies and made a miniprep of them. 2 out of the 8 samples contained the plasmid which I wanted (~6 kb).
When I run these samples on gel, I get more bands in the non-digested wells. Despite this, in case of single-digestion I clearly got the right size of the band and not the other bands. Furthermore, my DD is also OK!
Hi Norbert !
Non-digested plasmids do run at different sizes because of their topology or level of coiling. The proper way to test the length of a plasmid is the single digestion as you did. Remember also that the DNA in your ladder is linear.
Based on your wells SD4 to DD5, both your plasmids preps seem to be perfectly clean.
For more info, the supercoil question has been addressed here before (see link)
Nico
PS: the keyword "colonialism" is I think inappropriate here ;-) (see second link)
https://www.researchgate.net/post/Does_supercoiled_dna_migrate_faster_in_agarose_gel_electrophoresis_than_linear_form_of_dna
https://en.wikipedia.org/wiki/Colonialism
Hi Nicolas! Thanks for reply! Now I can do my midiprep and sequencing.
Btw, colonialism was automatically added by RG :)
Thanks again!
Hi,
Agreed: you cannot read the size of ND4 and ND5 based on their position (single cut is needed). Also, you could try colony-PCR (see link ;) ) as a 'quick-and-dirty' way to check your inserts straight from the plate.
P
http://openwetware.org/wiki/Endy:Colony_PCR
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